Methods and compositions for promoting immunopotentiation

ABSTRACT

This invention discloses immunopotentiating agents which stimulate an immune response. These agents are categorized into single agents that act directly, adjuvants added concurrently with the agents, or heteroconjugates. Heteroconjugate agents elicit or enhance a cellular or humoral immune response which may be specific for an epitope contained within an amino acid sequence. Enhanced hematopoieses by bone marrow stem cell recruitment was also a result of administering some of these agents. Examples of immunopotentiating agents include monoclonal antibodies and proteins derived from microorganisms (e.g., enterotoxins) which activate T cells. One method of treatment disclosed uses only the immunopotentiating agent to stimulate the immune system. Another uses adjuvants in combination with the agent. A third method employs heteroconjugates. Heteroconjugates comprise: (a) an immunopotentiating protein which is characterized as having an ability to stimulate T cells; and (b) a second protein having an amino acid sequence which includes an epitope against which a cellular or humoral response is desired. This invention also relates to a method of preparing the heteroconjugate, and to a method of stimulating the immune system in vivo in a novel way. One route of stimulation is to activate T cells, in some instances, specific subsets of T cells, by administering heteroconjugates containing an immunopotentiating protein and a second protein, to mammals. For this method of treatment, the second protein in the heteroconjugate is derived from abnormal or diseased tissue, or from an infectious agent; alternatively, the second protein is produced synthetically by standard methods of molecular biology. Sources of the second protein include tumors, viruses, bacteria, fungi, protozoal or metozoal parasites. Monoclonal antibodies or T cells prepared from mammals whose immune systems have responded to administration of the heteroconjugate may be produced and administered to induce passive immunity. A method of preparing a hybridoma which secretes said monoclonal antibodies and use of these monoclonal antibodies and T cells, are also disclosed. This invention is also directed to a vaccine comprising the heteroconjugate.

The government may own certain rights in the present invention pursuantto NIH grant number 5 RO1-CA-49260.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to immunology, and, more specifically, to thepreparation and use of immunopotentiating agents which are capable ofeliciting, enhancing and/or otherwise modifying immune responses. Theseagents, through their ability to elicit or enhance cellular or humoralresponses, have potential utility in a variety of disease conditionswherein immunotherapy might be expected to provide a benefit.

2. Description of Related Art

The body's immune system serves as a defense against a variety ofconditions, including, e.g., injury, infection and neoplasia, and ismediated by two separate but interrelated systems, the cellular andhumoral immune systems. Generally speaking, the humoral system ismediated by soluble products, termed antibodies or immunoglobulins,which have the ability to combine with and neutralize productsrecognized by the system as being foreign to the body. In contrast, thecellular immune system involves the mobilization of certain cells,termed T-cells, that serve a variety of therapeutic roles.

(a) The Immune System

The immune system of both humans and animals include two principalclasses of lymphocytes: the thymus derived cells (T cells), and the bonemarrow derived cells (B cells). Mature T cells emerge from the thymusand circulate between the tissues, lymphatics, and the bloodstream. Tcells exhibit immunological specificity and are directly involved incell-mediated immune responses (such as graft rejection). T cells actagainst or in response to a variety of foreign structures (antigens). Inmany instances these foreign antigens are expressed on host cells as aresult of infection. However, foreign antigens can also come from thehost having been altered by neoplasia or infection. Although T cells donot themselves secrete antibodies, they are usually required forantibody secretion by the second class of lymphocytes, B cells.

There are various subsets of T cells, which are generally defined byantigenic determinants found on their cell surfaces, as well asfunctional activity and foreign antigen recognition. Some subsets of Tcells, such as CD8⁺ cells, are killer/suppressor cells that play aregulating function in the immune system, while others, such as CD4⁺cells, serve to promote inflammatory and humoral responses. (CD refersto cell differentiation cluster; the accompanying numbers are providedin accordance with terminology set forth by the International Workshopson Leukocyte Differentiation (5). A general reference for all aspects ofthe immune system may be found in (1)).

(b) T Cell Activation

Human peripheral T lymphocytes can be stimulated to undergo mitosis by avariety of agents including foreign antigens, monoclonal antibodies andlectins such as phytohemaggluttinin and concanavalin A. Althoughactivation presumably occurs by binding of the mitogens to specificsites on cell membranes, the nature of these receptors, and theirmechanism of activation, is not completely elucidated. Induction ofproliferation is only one indication of T cell activation. Otherindications of activation, defined as alterations in the basal orresting state of the cell, include increased lymphokine production andcytotoxic cell activity.

T cell activation is an unexpectedly complex phenomenon that depends onthe participation of a variety of cell surface molecules expressed onthe responding T cell population (2, 3). For example, theantigen-specific T cell receptor (TcR) is composed of a disulfide-linkedheterodimer, containing two clonally distributed, integral membraneglycoprotein chains, αand β, or γ and δ, non-covalently associated witha complex of low molecular weight invariant proteins, commonlydesignated as CD3 (the older terminology is T3) (2, 4).

The TcR α and β chains determine antigen specificities (6). The CD3structures are thought to represent accessory molecules that may be thetransducing elements of activation signals initiated upon binding of theTcR αβ to its ligand. There are both constant regions of theglycoprotein chains of TcR, and variable regions (polymorphisms).Polymorphic TcR variable regions define subsets of T cells, withdistinct specificities. Unlike antibodies which recognize soluble wholeforeign proteins as antigen, the TcR complex interacts with smallpeptidic antigen presented in the context of major histocompatabilitycomplex (MHC) proteins. The MHC proteins represent another highlypolymorphic set of molecules randomly dispersed throughout the species.Thus, activation usually requires the tripartite interaction of the TcRand foreign peptidic antigen bound to the major MHC proteins.

With regard to foreign antigen recognition by T cells the number ofpeptides that are present in sufficient quantities to bind both thepolymorphic MHC and be recognized by a given T cell receptor, thusinducing immune response as a practical mechanism, is small. One of themajor problems in clinical immunology is that the polymorphic antigensof the MHC impose severe restrictions on triggering an immune response.Another problem is that doses of an invading antigen may be too low totrigger an immune response. By the time the antigenic level rises, itmay be too late for the immune system to save the organism.

The tremendous heterogeneity of the MHC proteins among individualsremains the most serious limiting factor in the clinical application ofallograft transplantation. The ability to find two individuals whose MHCis identical is extremely rare. Thus, T cells from transplant recipientsinvariably recognize the donor organ as foreign. Attempts to suppressthe alloreactivity by drugs or irradiation has resulted in severe sideeffects that limit their usefulness. Therefore, more recent experimentaland clinical studies have involved the use of antibody therapy to alterimmune function in vivo. The first successful attempt to develop a moreselective immunosuppressive therapy in man was the use of polyclonalheterologous anti-lymphocyte antisera (ATG) (7, 8, 9).

Clinical trials of the ATG treatment suggested a significant reductionof early rejection episodes, improved long term survival and, mostimportantly, reversal of ongoing rejection episodes. However, theresults were often inconsistent due to the inability to standardizeindividual preparations of antisera. In addition, the precise nature ofthe target antigens recognized by the polyclonal reagents could not bedefined, thus making scientific analysis difficult. The advent ofmonoclonal antibody (mAb) technology provided the basis for developingpotentially therapeutic reagents that react with specific cell surfaceantigens which are involved in T cell activation.

(c) Effect of Monoclonal Antibodies on the Immune System

Monoclonal antibodies (mAb) were developed by Kohler and Milstein in1975. The methods generally used to produce mAb consist of fusing(hybridizing) two types of somatic cells: (1) a neoplastic myeloma cellline; and (2) a normal B lymphocyte obtained from an immunized animal.The result is called a hybridoma which is characterized by immortalgrowth and the ability to secrete antibodies specific for theimmunization antigen.

One of the clinically successful uses of monoclonal antibodies is tosuppress the immune system, thus enhancing the efficacy of organ ortissue transplantation. U.S. Pat. No. 4,658,019, describes a novelhybridoma (designated OKT3) which is capable of producing a monoclonalantibody against an antigen found on essentially all normal humanperipheral T cells. This antibody is said to be monospecific for asingle determinant on these T cells, and does not react with othernormal peripheral blood lymphoid cells. The OKT3 mAb described in thispatent is currently employed to prevent renal transplant rejection (10).

One unexpected side effect of the OKT3 therapy was the profoundmitogenic effect of the mAb in vivo (28). Although anti-CD3 mAb has beenshown to activate T cells in vitro to produce various lymphokines, etc.(11), OKT3 has not been previously used to stimulate the immune systemin vivo.

In addition, other cell surface molecules have been identified that canactivate T cell function, but are not necessarily part of the T cellsurface receptor complex. Monoclonal antibodies against Thy-1, TAP,Ly-6, CD2, or CD28 molecules can activate T cells in the absence offoreign antigen in vitro (12, 13, 14, 15, 16). Moreover, certainbacterial proteins although differing in structure from mAbs, also havebeen shown to bind to subsets of T cells and activate them in vitro(17). Although some of these agents, in vitro effects have previouslybeen demonstrated, in vitro activity is often not a reliable predictorof in vivo effects.

(d) Immune System and Tumor Growth

One cause of malignant tumor growth is believed to be the inability ofthe immune system to respond appropriately to tumor antigen. Forexample, malignant progressor tumors are only weakly immunogenic and canevade host recognition and rejection. Both specific and non-specificeffector pathways have been implicated in tumor immunity. Treatment byimmunotherapy is aimed at remedying defects in the immune weaponry. Theaim of immunotherapy has been the enhancement of one or both of thesepathways. One potential approach to therapy is to activate hostantitumor cellular effector mechanisms.

Historically, non-specific adjuvants such as BCG or pertussis have beenused to augment immune responses. In normal individuals these adjuvantsamplify immune responses by providing non-specific stimuli that enhanceoverall immunity. However, these adjuvants do not selectively act on Tcells, or subsets of T cells, and have not been shown capable ofovercoming immunodeficiency states. Unfortunately, current modes ofimmunotherapy which induce non-specific effector cells are not effectiveenough in potentiating anti-tumor responses (18). Recently,immunotherapy regimens which utilize the ability of the immune system torecognize tumor antigens in a specific manner, for instance utilizingspecific tumor-infiltrating lymphocytes, for immunotherapy, have beensuggested to result in superior anti-tumor immunity (19). Thus, currentefforts toward developing more efficacious forms of immunotherapy havefocussed on specific anti-tumor response and memory-following antigenrecognition. One approach that has not previously been accomplished hasbeen the in vivo administration of T cell activating mAbs to promoteanti-tumor activity.

(e) Defenses Against Viral Infections

Human immunodeficiency virus (HIV), the biologic agent of AIDS, causes apersistent infection associated with profound immunosuppressionresulting in susceptibility to opportunistic infections. Immunologicalresponses to HIV infection require the development of both humoral andcell mediated effector mechanisms; however current efforts in treatmentand vaccine design have fallen short of success either due to theimmunodeficiency associated with the viral infection, or to the lowimmunogenicity of the vaccine (20). The development of a safe andeffective vaccine against infection with human immunodeficiency virus(HIV) is complicated by a lack of understanding of protective immunityto HIV and disease development, and the absence of an adequate andconvenient animal model for studying HIV infection.

Because HIV can be transmitted as either a cell-free or cell-associatedvirus, a protective immune response against HIV infection will likelyrequire both humoral and cell-mediated immunity, including neutralizingantibody against HIV, antibodies involved in antibody-dependent cellularcytotoxicity and cytotoxic lymphocytes. All of these activities involvevirus-specific T cells. T cell activation requires potent in vivo immuneresponses to foreign antigens such as viruses.

In individuals infected with HIV, two components of the immune systemare suboptimal and, therefore, the ability to generate an immuneresponse in these individuals has been compromised. First, the reducedfrequency of antigen-reactive CD4⁺ T cells is apparently not sufficientto mount an appropriate immune response to HIV, especially if thequantity of HIV antigen is low. CD4 is a membrane protein that acts as abinding site and entry port into CD4⁺ lymphocytes for the humanimmunodeficiency virus-type 1 (HIV-1) (21). Second, all immune responsesare dependent on the ability of T cells to recognize processed antigenassociated with major histocompatibility antigens (MHC). Any vaccineapproach which utilizes HIV peptides or inactivated virus antigen mustdepend on the ability of antigenic peptides to bind the appropriate MHCantigens necessary to initiate an immune response. Given the tremendouspolymorphism of the MHC antigens expressed in the population, and thevariation of the HIV virus, developing a successful HIV vaccine forgeneral use is difficult and has not yet been successful.

(f) Problems in Developing Vaccines to Weakly Immunogenic Antigens

The usefulness of certain peptides, proteins or other potential ordesired immunogens in vaccines can be limited by several criticalfactors. For example, low immunogenicity of the peptide or otherstructure which one desires to employ can be a difficult problem toovercome, particularly with smaller peptides and those peptides which donot contain appropriately strong B- and/or T-cell potentiatingsequences. Such peptides are typically only weakly immunogenic at best.Moreover, to be of widespread applicability, the peptides chosen must becapable of inducing an immune response in a majority of the population.

It has been difficult to protect against attack by organisms such as theHIV virus or to provide tumor immunity for several reasons. For example,genetic differences exist among individuals at the majorhistocompatibility locus, which limits the system's ability to respondto individual small peptides. Thus, the various components of the immuneresponse, including the T cells and B cells, may not interactappropriately in generating a response to non- or weakly-immunogenicsmall peptides. Attempts to improve peptidyl immunogenicity havecentered principally on the use of adjuvants such as alum or completeFreund's adjuvant. However, prior adjuvants such as these have proven tobe inadequate for various reasons, including an inability of theadjuvant to specifically enhance T or B cell activity and the inabilityof the adjuvant to overcome the severe limitations of MHC restriction.

Although glimpses into the defense mechanisms of the body's own immunesystem have been provided by in vitro studies and by observation of somein vivo reactions, there is a serious lack of successful therapeuticmethods to augment immunity in vivo. Improved compositions and/ormethodology for eliciting or enhancing cellular or humoral responses inmammals are needed both to provide animal models for investigation oftherapeutic regimes, to provide novel means of preparing improved immunesystem-directed products such as improved immunotherapeutic antibodies,and to advance treatment and possible immunization, e.g., for conditionssuch as HIV, cancer and infections.

SUMMARY OF THE INVENTION

The present invention is concerned with a broad array of embodiments,generally involving methods and compositions for potentiating one ormore aspects of the immune response of a human or other animal having animmune system, as well as to products which may be derived out of theuse of these methods and compositions. Generally speaking, the inventionconcerns essentially four categories of what may be referred to broadlyas immunopotentiating or immunoactivating compositions: 1) individualimmunopotentiating agents which are used to potentiate one or moreaspects of the immune system; 2) immunopotentiating “adjuvant”compositions wherein immunopotentating agents are employed essentiallyas “adjuvants” to improve the body's immune responsiveness to othercompounds which are co-administered with, or included in with,compositions containing the immunopotentiating agent; 3)immunopotentiating conjugates wherein the immunopotentiating agent isactually chemically coupled to the compound against which an immuneresponse is desired; and 4) products derived from the administration ofone or more of the foregoing, including, e.g., antibodies,antibody-producing cells, T-cells, potentiated bone marrow progenitorcells, and the like.

The term “activation” is generally defined to refer to any changeinduced in the basal or resting state of T or B cells. This includes,but is not limited to, increased cell proliferation and DNA synthesis,lymphokine and cytotoxic cell production, a rapid rise in intracellularcalcium, release of water soluble inositol phosphates, increased IL-2receptor expression, enhanced proliferative response to IL-2, andenhanced responses to foreign antigens or MHC (23). In contrast, theterm “immunopotentiating” is classically defined as the ability toproduce an effect on the immune system which enhances the system'sability to respond to foreign antigens. Thus, immunopotentiation mayaffect the cellular response, humoral response, or both. Exemplaryindices of immunopotentiation include, but are not limited to,cell-proliferation, increased DNA synthesis, increased production oflymphokines, increased production of cytotoxic cells, calcium efflux, orany other change that raises the cell above the basal or resting state.

While one can consider there to be a distinction between the terms“potentiation” and “activation”, in the context of the present inventionthe use of the term “potentiation” is intended to include bothpotentiation and activation. Thus, the immunopotentiation achieved bythe methods and agents of this invention may affect all T cells, certainsubsets of T cells, or B cells, depending on the nature of the agent(s)and their dose levels. One of the objects of this invention is toprovide means for fine tuning immunopotentiation, allowing one to targetT and/or B cell response depending on the nature of the clinicalcondition to be treated.

Accordingly, in certain general embodiments, the present inventionconcerns the preparation and use of immunopotentiation agents, whetherused alone as a direct immunopotentiation agent, or combined with othercompounds, either covalently or simply admixed in the same composition.In the context of the present invention, the term immunpotentiationagent is intended to include immunopotentiating antibodies, as well ascertain bacterial proteins which have been determined to have profoundimmunopotentation actions.

In terms of antibodies, useful immunopotentiation agents will generallyinvolve antibodies against a cell surface epitope of T-cells whereinbinding of the antibody to the surface epitope of the T-cell will resultin immunopotentiation. An exemplary antibody is anti-CD3 (e.g., OKT3),previously known only to be immunosuppressive and not previously knownto be immunopotentiating. The present inventor has surprisinglydiscovered that, in fact, when anti-CD3 is administered at relativelylow doses (e.g., on the order of 100 to 200 μg/kgm body weight), ratherthan being immunosuppressive it exhibits a very profoundimmunopotentiation effect. The reason for this appears to include butmay not be limited to the induction of lymphokines, the proliferation ofT cells, or even the progression of T cells from a naive to memorystate.

While anti-CD3 is a useful immunopotentiation agent, numerous otherimmunopotentiation antibodies are contemplated to be within the scope ofthe present invention. Such antibodies are defined generally asantibodies which recognize and activate a T cell activation molecule orepitope on the cell surface of T cells. For example, monoclonalantibodies which are particularly useful in the practice of the presentinvention will comprise those directed against the T cell variable orconstant epitopes on the cell surface of T cells. The T cell activationmolecules which are expressed on the cell surface may be either thoseassociated with the T cell receptor complex or those with the antigensdistinct from, that is not physically associated with, TcR on the cellsurface. Specific embodiments of T cell activation molecules compriseeither the variable or the constant region epitopes as expressed on theantigen specific T cell receptor polymorphic chains, e.g., α, β, γ, andδ chains.

Embodiments of the non-polymorphic TcR associated CD3 chains againstwhich monoclonal antibodies may be directed for use asimmunopotentiating agents are the γ, δ, ε or ζ chains. Monoclonalantibodies have been developed against some of these chains, asexemplified by OKT3, SP34, UCHTI or 64.1 (68-70). Among the T cellsurface antigens which are distinct from, and not physically associatedwith, TcR, are CD2, CD28, Thy-1, and the activation epitopes expressedon members of the Ly-6 protein family.

As noted, the immunopotentiation agents of the present invention willalso include certain potentiating bacterial proteins such as bacterialenterotoxins, exemplified by staphylococcal enterotoxin B (SEB). SEB isnow known to activate T-cells and provide surprisingly profound andsubset-specific potentiation. As with some of the potentiatingantibodies, the mechanism of how enterotoxins function to stimulate theimmune system is not entirely clear, but could involve lymphokineproduction or T cell proliferation. While SEB comprises a preferredenterotoxin for immunopotentation purposes, the invention contemplatesthat other similar enterotoxins, such as staphylococcal enterotoxins A,C₁, C₂, D, E, toxic shock syndrome toxin (TSST), exfoliating toxin(ExFT) and likely even mycoplasma arthriditis substance, will findsimilar utility.

For use directly as immunopotentiation agents, one will generally desireto first combine the agent in a pharmacologically suitable vehicle, suchas combining with an appropriate diluent or buffer, in an amount andconcentration that is appropriate to effectuate potentiation of theimmune system when administered. Typically, in the case of, e.g.,anti-CD3, one will desire to provide parenteral compositions having fromabout 0.1 mg/ml to about 1 mg/ml, in order to allow the parenteraladministration of appropriate amounts of the antibody.

As mentioned, the present invention contemplates the use of theseimmunopotentiating agents in immunogen containing compositions such asvaccines, where the agents serve as “adjuvants” to improve theimmunogenicity of other components of the composition. Thus, it iscontemplated that through the use such agents as “adjuvants”, thepreparation of useful vaccines using only weakly or non-immunogenicmolecules not previously known to function as immunogens is enabled. Insuch embodiments, the immunopotentiating protein is admixed or otherwiseco-administered with the molecule against which an immune response isdesired, with the immunopotentiating agent being present in amountseffective to promote potentiation upon administration to the particularsubject.

It is proposed that still further advantages will be realized where theimmunopotentiation agent is actually conjugated to the molecule againstwhich an immune response is desired. These conjugates are referred to inthe context of the present invention as “heteroconjugates”. As usedherein, an immunopotentiating heteroconjugate is defined as animmunopotentiating agent conjugated to a second protein or othermolecule against which an immune response is desired, the conjugationbeing in the nature of a chemical or molecular crosslink.

Exemplary embodiments of the second molecule include proteins whichcomprise amino acid sequences or other potential determinants (e.g., anon-amino acid determinant such as a sialo group) against which acellular or humoral immune response is desired. However, as with theadjuvant embodiments, the present invention also contemplates the use ofnon-protein molecules, such as glycolipids, carbohydrates or evenlectins, as the second molecule against which an immune reaction issought. All that is required for use in the context of heterconjugatesis that one be capable of conjugating the molecule to theimmunopotentiation agent.

The immunopotentiating protein of the heteroconjugate may also be formedby linking two monoclonal antibodies directed against two distinct butspecific T cell epitopes. A specific embodiment of this type ofimmunopotentiating protein is anti-CD3 coupled with anti-CD4 to form aCD4 subset-specific immunopotentiating protein. It has been demonstratedthat administering of such a bispecific antibody construct in vivoactivates T cells to a surprising degree (see, e.g., U.S. Pat. No.4,676,980 to Segal, and FIG. 14 herein). It is proposed by the presentinventor that one may employ such a bispecific antibody construct as theimmunopotentiating ligand of heteroconjugates formed with a secondprotein against which an immune response is desired.

Use of the heteroconjugate disclosed in this invention is believed toaid in directing the immune attack to specific epitopes, via theattached compound or protein. Thus, it is proposed that a particularutility of these embodiments will be found in the treatment of diseasessuch as cancer, where only weakly immunogenic tumor-specific ortumor-associated proteins are found to appear on the cell surface ofmany tumors, or are found to characterize the tumor.

It is proposed that other particularly important applications are in thecontext of viral-specific or viral-associated epitopes, particularlythose where the immune system may be compromised, such as in the case ofHIV infections. Moreover, amino acid sequences homologous to thosederived from genes in a bacteria, fungus, protozoal or metazoalparasite, may also be used as conjugating agents.

Of course, where desired, the epitopes used as second proteins mayeither be derived from the cell surface or extracted from cells whichare infected or diseased. Alternatively, amino acid sequences can bemade synthetically by standard chemical synthetic or molecular biologymethods. Thus, for example, in the case of HIV immunotherapy, amino acidsequences are known for various peptides isolated from the HIV virusgp120 cell membrane which can be employed as embodiments of the secondcompound, whether it be conjugated to the immunopotentiating protein orsimply admixed in an appropriate pharmaceutical vehicle. Preferredembodiments of peptides which may be used as second proteins in aheteroconjugate used to augment immunity against HIV, comprise peptides18, T1, T2, or peptides derived from the CD4 binding site.

In similar fashion, epitopes specific for amino acid sequenceshomologous with those expressed on the surface of human-hepatitis virus,or extracted from cells infected with the virus may be used as thesecond protein. Viral specific epitopes may also comprise aminosequences homologous with those expressed on the surface of infectedcells.

Immunogenic compositions may be prepared for administration to subjectsby including any of the foregoing immunopotentiating compositions, andformulated to include an effective amount of 1) an immunopotentiatingprotein (i.e., where one desires simply generalized immunopotentiationrather than potentiation that is directed against a second compound), 2)an immunopotentiation protein in combination with a second compound, or3) an immunopotentiation protein conjugated to such a second compound.The materials that are formulated are preferably renderedpharmacologically acceptable by manufacturing in accordance with goodmanufacturing practices, extensively dialyzed to remove undesirablesmall molecular weight molecules and/or lyophilized for more readyformulation into a desired vehicle.

Where such compositions are intended for human administration, one willtypically desire to include an amount of immunopotentiation agent thatwill result in T cell activation in the absence of concommitantimmunosuppression. The determination of exact amounts will depend on theparticular circumstances, such as the particular condition to betreated, the physical condition of the patient, the type of immunogeniccomposition that is to be administered, and the like. Thus, e.g., basedon murine studies performed by the inventor one can extrapolate in thecase of embodiments incorporating immunopotentiating antibodies such asanti-CD3, anti-CD28 or anti-CD2, that suitable formulations shouldtypically include from about 10 ug to about 1000 ug bolus/patient every14 days or so, and more preferably 100 ug to about 400 ug of theantibody per patient. Similarly, one can extrapolate in the case ofenterotoxins that one should typically desire to employ from about 100ug to about 10 mg of the enterotoxin per dosage, and more preferablyabout 1 to about 5 mg/dose.

For the preparation of compositions suitable for parenteraladministration, the immunogens of the invention may be formulated inoils such as sesame or peanut oil, aqueous propylene glycol, inliposomes or in sterile aqueous solutions. Such solutions are typicallysuitably buffered if necessary and the liquid diluent first renderedisotonic with sufficient saline or glucose. Additionally, stabilizers inthe form of, for example, sorbitol or stabilized gelatin may beincluded. These particular aqueous solutions may be particularly wellsuited for intramuscular and subcutaneous injections, as may bepreferred for vaccination using antigenic preparations.

The proteins may be formulated into the composition as neutral or saltforms. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the peptide) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isoprophylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The compositions are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. Suitable regimens for initialadministration and booster shots are also variable, but are typified byan initial administration followed by subsequent inoculations or otheradministrations.

Various methods of achieving additional adjuvant effect for the vaccineincludes use of agents such as aluminum hydroxide or phosphate (alum),commonly used as 0.05 to 0.1 percent solution in phosphate bufferedsaline, admixture with synthetic polymers of sugars (Carbopol) used as0.25 percent solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between 70° to 101° C. for 30 secondto 2 minute periods respectively. Aggregation by reactivating withpepsin treated (Fab) antibodies to albumin, mixture with bacterial cellssuch as C. parvum or endotoxins or lipopolysacchraide components ofgram-negative bacterial, emulsion in physiologically acceptable oilvehicles such as mannide mono-oleate (Aracel A) or emulsion with 20percent solution of a perfluorocarbon (Fluosol-DA) used as a blocksubstitute may also be employed.

While administration of the foregoing immunopotentiating compositionswill likely find their greatest utility and application in the treatmentof human disease, the invention is by no means limited to humanapplication, and is intended to apply to any mammal having an immunesystem, including, e.g., rodents such as mice, hamsters and rats,primates, rabbits, even farm animals such as cows, pigs, etc. Thus, theinvention contemplates, e.g., that certain of the foregoing embodimentswill have general applicability wherever one desires to obtain anenhanced immune response against a desired molecule, such as in theinitial immunization of animals for hybridoma or even polyclonalantibody development.

While it is contemplated that the nature of the second molecule is notcrucial to the successful practice of the invention, it is recognizedthat the invention will find its greatest utility where the secondmolecule is a peptide, in that peptides are often notoriously difficultto obtain an immune response against. Thus, it is believed thatparticular benefits will be realized through the use of peptides havingfrom about 8 to about 100 amino acids in length, and even morepreferably, about 8 to about 50 amino acids in length.

In the context of heteroconjugates, it is contemplated that numerousmethods for conjugation may be applied, including but not limited to: 1)the formation of biotin-avidin bridges; 2) the use of cross linkers suchas SPDP to link the functional units; 3) cross-linking of maleimide andSH groups; as well as numerous other possibilities. In general, all thatis required is that the cross-linking maintain the integrity of thepeptidic antigen and leave unaltered the activating property of theimmunopotentiating reagent.

As mentioned above, the present invention contemplates that varioususeful biological products may be derived through the application of theforegoing immunopotentiating compositions. For example, the adjuvant andheteroconjugate embodiments will provide extremely useful means forpreparing antibodies, including monclonal antibodies. Moreover, it hasbeen found that immunopotentiating antibodies such as anti-CD3 can serveto promote the recruitment of hematopoetic progenitor cells, presumablyby stimulating the release of cytokines and lymphokines from activatedT-cells. This lends the possibility that such embodiments may beemployed to prepare highly active bone marrow for transplantation, oreven for administration to bone marrow transplant recipients or thosewith depleted bone marrow cells to provide a metabolic boost to themarrow. Moreover, it is contemplated that activated T cells themselveswill find some utility, such as in anti-tumor therapy that employstumor-infiltrating lymphocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention will become apparent uponreading the following detailed description and upon reference to thedrawings in which:

FIG. 1. Forms of Immune Augmentation. This figure demonstrates anoverview of various embodiments of the invention, including“immunoadjuvant” embodiments wherein the immunopotentiating protein issimply admixed with a compound against which an immune response isdesired (top panel), or where and actual heteroconjugate is formedbetween the immunopotentiating protein and the compound (middle panel).In the bottom panel is shown an embodiment wherein theimmunopotentiating ligand is actually a bifunctional cojugate formedbetween two antibodies.

FIG. 2. Activation of peripheral lymph node T cells fromanti-CD3-treated C3H mice as assessed by flow cytometry (FCM). Two colorFCM from control animals and those treated with, 4, 40, or 400 μg ofanti-CD3 are displayed as contour plots on a logarithmic scale.Intensity of green FITC fluorescence is plotted along the x-axis and red(B-phycoerythrin) fluorescence is plotted along the y-axis. (A) Anti-CD4staining on the x-axis and anti-IL-2 receptor (Il-2R) staining on they-axis. (B) Anti-CD3 staining on the x-axis and anti-Thy-1 staining onthe y-axis. C3H/HeN MTV⁻ mice were killed 18 hours after intravenousinjection of purified anti-CD3 (MAb 145-2C11) that was grown andpurified as described (24). Femoral, axillary, and mesenteric lymphnodes were removed and dissociated into a single-cell suspension and FCManalysis was performed (25). Cells were stained with FITC-anti-CD3 orFITC-anti-CD4 (MAb GK1.5) (Becton Dickinson), and biotin-conjugatedanti-IL-2R (MAb 3C7) or biotin-conjugated MAb to Thy-1.2 (BectonDickinson), then counterstained with B-phycoerythrin-conjugated eggwhite avidin (Jackson Immuno Research Laboratories). These results showthat low dose (4 ug) anti-CD3 treatment activates T cells as evidencedby IL-2R expression but does not modulate T cell receptors.

FIG. 3. In vitro proliferation of lymph node T cells from anti-CD3treated C3H mice. Eighteen hours after intravenous anti-CD3administration, lymph nodes were removed from animals and the cells(1×10⁵ cells for mixed lymphocyte reaction (MLR) and mixed lymphocytetumor culture (MLTC)) were incubated in medium that contained irradiatedsyngeneic spleen cells (2×10⁵) plus recombinant IL-2 (rIL-2), orirradiated allogeneic (C57BL/10) spleen cells, or mitomycin C-treatedPro-4L tumor cells (5×10³). Proliferation was measured by [³H]thymidineuptake at 48 hours (rIL-2) or 72 hours (MLR and MLTC). Background uptake(generally less than 5×10³ cpm) was determined from lymph node cellsstimulated with syngeneic irradiated accessory cells, or from mitomycinC-treated tumor cells cultured alone, and was subtracted from values fortreated cells. All assays were performed in triplicate; standarddeviations were less than 5%. The results show that a) functional IL-2Ris expressed on anti-CD3 treated cells; and b) anti-CD3 treated T cellsresponded more vigorously to allogeneic MHC and tumor antigens than diduntreated cells under otherwise similar conditions.

FIG. 4. Colony Stimulating Factor (CSF) in serum of mice after injectionof anti-CD3. Pooled sera from three animals were placed at 6% finaldilution with murine bone marrow cells, and the number of colonies werecounted after 7 days. Each sample was tested in duplicate and theresults were averaged. In all cases, duplicate values differed by nomore than 5% A: Mice received 400 μg anti-CD3 Ig(▪), or 250 μg ofF(ab′)₂ fragments of anti-CD3 (∘). (The number of colonies at 3 h foranti-CD3 treated mice was more than 300.) B: Number of colonies aftervarious doses of anti-CD3. Serum was collected 3 h after injection.These results show that anti-CD3 in vivo induces lymphokines includingcolony stimulating factors.

FIG. 5. Clinical Response to anti-CD3 (OKT3) Treatment. A: Increasedallogenic MHC response in patients treated with OKT3. B: Proliferationof T cells before and after OKT3 treatment in the presence(cross-hatched bars) or absence (closed bars) of rIL-2 suggest that invivo treatment with OKT3 activates human T cells.

FIG. 6. Anti-CD3 augments immune response. Anti-KLH (Keyhole limpethemocyanin) antibodies in mice treated with KLH using PBS, CFA, oranti-CD3 as immunoadjuvants. The results suggest anti-CD3 potentiatesanti-KLH antibody responses.

FIG. 7. Alloresponse of lymphocytes from C3H mice treated withStaphylococcus enterotoxin B (SEB) or mAb 145-2C11. The response asmeasured by cell proliferation (3H uptake in CPM) was compared amongcontrol cells versus those treated either with mAb 145-2C11 (anti-CD3)or one of 3 doses of SEB. Cell proliferation was increased above controllevels by either treatment. In addition, the stimulation by SEB showed adose response.

FIG. 87. IL-2R expression on T cells from SEB-treated mice. Mice weretreated with increasing doses of SEB (0, 5, 50, 250 μg). Il-2Rexpression after 18 hours was compared using flow cytometry and showedenhanced expression. Dose response was observed.

FIG. 9. Proliferative response to SEB. Lymph node cell proliferation wasassayed by ³H uptake (CPM) in C3H mice at 18 hours after administrationof SEB. rIL-2 response to SEB ³H was enhanced compared to controls or to145-2C11-treated mice. In addition, there was a dose response (5, 50,250 μg of SEB).

FIG. 10. Expansion of V_(β)8⁺ cells in SEB-treated mice. Three daysafter treatment of mice with SEB, spleen cells were incubated withanti-V_(β)8 and V_(β)8⁺ cells were assayed by flow cytometry. Expansionof V_(β)8⁺ cells was observed due to SEB treatment.

FIG. 11. Distribution of lymphocyte subsets after treatment with lowdoses of OKT3. After treatment with low dose OKT3 (100 μg/patient),there was observed an initial decrease in lymphocyte subsets, followedby a dramatic recovery of CD2, CD3 and CD4 positive cells, and to alesser extent CD8 positive lymphocytes. In addition, cells were observedto be activated by the anti-CD3 treatment based on an enhancedexpression of IL-2 receptor (CD25) on post-treatment lymphocytes.

FIG. 12. Increased lymphokine production after treatment of humans withOKT3. In response to in vivo treatment of a patient with a 100 μg doseof OKT3, relative levels of hematopoetic progenitor cells (e.g., bands,myelocytes) are seen to increase dramatically in response to OKT3administration.

FIG. 13. Survival of grafts as a function of treatment with anti-CD3. Inthe study reflected by this figure, the ability of anti-CD3 to abrogategraft versus host disease (GVHD) was tested in a murine model whereinthe mAb treatment also enhanced bone marrow engraftment. The study wascarried out as follows: (B10×B10.BR)F₁ mice were sublethally inradiatedwith 500 RADs and injected with a lethal dose of GVH reactive B10.BRspleen cells in the presence or absense of immunosuppressive anti-CD3.The results, as displayed in the figure, show that when a controlantibody was administered (normal hamster Ig), there was no survival byday 13. However, at the same time point, survival was about 25% forlower dose administration of anti-CD3 (25 μg) and a remarkable 75% atthe higher dose (250 μg) suggesting the anti-CD3 had prevented graft v.host disease. Median survival was 11 days in controls and 12 days inanimals treated with the low-dose of anti-CD3 (25 μg). Survival in thehigh dose anti-CD3 group plateaued at 75%.

FIG. 14. Effects of treatment of mice in vivo with a combination of1005/45-KLH and 145-2C11. This figure reflects studies conducted todemonstrate the adjuvant characteristics of anti-CD3 (mAb 145-2C11, ananti-murine CD3 mAb specific for a 25 kd protein CD3-ε) whenco-administered with a KLH-linked peptide (1005/45; ref. 29), whenadministered intraperitoneally. C57BL/10 mice were administered 100 μgof the 1005/45-KLH conjugate, intraperitoneal (IP) or subcutaneously(SC), either alone or co-administered with 4 ug of anti-CD3 (145-2C11)IP. Titers of anti-1005/45 were assayed from 7-28 days post injection.The results demonstrate that when the peptide was administered alone byeither route, very little specific antibody titer developed. However,when anti-CD3 was co-administered, a surprising increase in specificantibody titer resulted.

FIG. 15. In vivo treatment with mAbs. The response of IL-2 receptorpositive cells was compared after treatment with anti-CD3 alone versusafter treatment with a heteroconjugate comprising anti-CD3 and anti-CD4.Anti-CD3 alone resulted in significant increases in both CD4+ and CD8+IL-2 receptor+ cells. The heteroconjugate (linked by the SPDP method)produced an enhanced-IL-2R expression on CD4+ cells, indicative of asubset-specific response.

FIG. 16. In vivo immune stimulation of mice administered aheteroconjugate. Panel A: IgG anti-FITC antibody production in anti-CD3treated mice immunized with FITC-BSA in complete Freunds adjuvant (CFA)or PBS, compared to ELISA measurements of sera from control mice (openbars). Panel B: IgG anti-FITC antibody production measured from sera ofFITC-anti-CD3 treated mice (left-hatched bars) compared to FITC-normalHamster Ig (cross-hatched bars) measured by ELISA performed on day 10bleeds.

FIG. 17. Two models of T:B collaboration. A) Direct T:B interaction. B)Classical MHC restricted antigen interaction. Panel A illustrates theconventional model of T-B collaboration namely that the ability of Tcells to recognize foreign antigen and promote immunoglobulin productiondepends on the recognition by TcR of peptidic antigen presented in thecontext of the polymophic MHC molecules. In panel B, it is proposed thatone can bypass the strict requirement of MHC-restricted antigenrecognition by the use of immunopotentiating hetercoconjugates. Forexample, F(AB′)₂ fragment of anti-CD3 are coupled to peptidic antigen tobridge the T cell with peptide specific B cells. The resultingcross-linking of the TcR by this T-B interaction would result in thelocalized secretion of helper factors necessary to augment B celltriggering and Ab production in the absence of an MHC-restrictedinteraction.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The body's immune system is not invincible. It succumbs to attack whenit is unable to recognize the need to repel invaders, for example, whenthe foreign or abnormal stimulus is too weakly immunogenic to produce aresponse. Genetic immunodeficiency states as well as acquiredimmunodeficiency (e.g., drug or viral induced) also undermine the immunesystem's ability to respond to infection or abnormal growth such asneoplasms. Moreover, artificial suppression of the immune system, oftenemployed in an attempt to minimize the risk of transplantation failuredue to host versus graft disease, is accompanied by the risk ofgenerating serious side effects, e.g., infection and even tumordevelopment. A different type of problem arises when non-selectivestimulation attacks normal as well as abnormal cells, leading toself-destruction.

That the immune system has a role in prevention or suppression ofmalignant growth is undisputed, but the nature and extent of that roleis unclear. Attempts have been made to strengthen the immune responseand/or to alter tumor responses to attack. However, such attempts todevelop suitable immunotherapeutic protocols have not generally beensuccessful. For example, both the transfer of immune lymphocytes as wellas immune stimulation has been employed in attempts to treat tumors incancer patients with little success. Moreover, the search for the “magicbullets,” such as through the injection of antibodies or of antibodieswith non-specific tumoricidal agents, e.g., toxins, have not resulted inthe hoped-for regression of tumors. Similarly, non-specific stimulationof the immune system of tumor-bearing individuals by injectingadjuvants, e.g., BCG, has met with only limited success (e.g., in thecase of melanoma). Moreover, immunization with unmodified allogenictumor cells is ineffective, and may even result in a more rapid tumorgrowth.

The present invention discloses a collection of related strategies forovercoming one or more failures of the prior art in immunotherapy. Thesuccess and potential success of this strategy is demonstrated in thefollowing examples. The strategy comprises producing newimmunopotentiating agents and compositions which employ, for example,the use of 1) selected immunopotentiating agents alone, 2) as“adjuvants” with second agents against which a selected response isdesired, or 3) as heteroconjugates wherein the immunopotentiating agentis conjugated to the second compound.

Shown in FIG. 1 is an overview of various embodiments of the invention.In the upper panel is shown a basic embodiment wherein animmunopotentiating protein (in the example shown, the immunopotentiatingprotein is an immunopotentiating antibody, anti-CD3) in an admixturewith a peptide against which an immune response is desired. In themiddle panel is shown a heteroconjugate embodiment, comprising aconjugate between an immunopotentiating protein (in this case anantibody) and a peptide against which an immune response is desired. Inthe bottom panel is shown a bifuncitonal ligand-conjugate formed betweentwo antibodies (e.g., anti-CD4 and anti-CD3), wherein one of theantibodies is coupled to a peptide against which an immune response isdesired.

As the following examples will illustrate, the success of the variousstrategies of the invention was demonstrated by in vivo responses ofanimals and humans to these immunopotentiating agents, indicating thatimmunopotentiation, tumor growth limitation, induced tumor immunity,resistance to infection, and enhanced bone marrow stem cell recruitment,has indeed been achieved as predicted by the inventor's novel theories.

1. Immunopotentation Agents Achieving Potentiation Directly

A. Protection Against Infection or Abnormal Growth (Neoplasm)

It has been shown in vitro that monoclonal antibodies (mAb) recognizingspecific T cell surface molecules can activate T cells in the absence ofnominal antigen (26, 27). The mAb apparently mimics the physiologicantigen and bypasses the T cell receptor (TcR) antigen-specificrecognition mechanism. While T cell activation is critical to generatingpotent in vivo immune responses to, for example, tumor antigens, in manyinstances, an individual's resident T cells are not sufficient to mountan appropriate immune response. To overcome this limitation, theinventor has developed in vivo treatments that are capable of enhancingthe immune potential of T cells, providing a powerful tool to amplifythe immune system.

Based on results illustrated in the following examples, it is now clearthat antibodies directed against TcR/CD3 and other T cell surfacestructures can profoundly potentiate T cell function in vivo. Thisaction of monoclonal antibodies was unexpected in vivo because a majorclinical use of mAbs directed against T cell surface proteins has beento suppress, not to stimulate, the immune response to foreign antigens.Moreover, monoclonal antibodies (mAb) to T lymphocyte antigens have beenused to suppress immune responses in vivo and in vitro by blocking Tcell receptor-mediated antigen recognition, a property exploitedclinically to prevent and reverse organ transplant rejection.

In the practice of the invention, an important aspect is the ability toidentify appropriate immunopotentiation agents which can be employed inimmunopotentation compositions hereof. This identification is routinelyaccomplished through the use of assays which identify potentiationand/or activation of immune system cells. For example, the inventorroutinely employs assays for activation of T cells such as might beexhibited as cytolytic activity by a responder cell such as activatedCD8+ cells, against a target cell such as K562 cells. Alternatively, Tcell activation can be read out by the production of lymphokines (e.g.,GMCSF, IL-2, -IFN) released by the responder cells. Such assays are wellknown in the art as illustrated by reference 64. Another useful assayemploys thymidine incorporation as a measure of T cell proliferation.(65). Other possible assays include IL-2 receptor expression,phosphoinositide turnover, and even Ca⁺⁺ flux (66, 67).

In the context of the present invention, the inventor routinely employsthe induction of cytolytic activity, lymphokine production and T cellproliferation as assays for immunopotentiation. Exemplary assays are setforth in Tables 1 and 2 below, which were employed to identify mAb145-2C11 (29), the mAb specific for the 25 kd protein CD3-ε. The dataset forth in these two tables together demonstrate the ability of the145-2C11 mAb to activate T cells. In Table 1, an assay is set forthwherein the ability of the mAb to redirect lysis of cytotoxic Tlymphocytes (CTLS) to irrelevant target cells is assessed. Details ofthe assay are as set forth in references 29 and 45. The resultsdemonstrated that none of the alloreactive murine CTLs significantlylysed the human target cell K562 in the absence of mAb (Table 1).However, addition of the culture supernatant from the hybridomasecreting anti-CD3-ε mAb resulted in lysis of target cells.

The lysis was dependent on the presence of effector CTLs, as incubationof the K562 cells with the mAb alone did not result in target-celllysis. None of several other antibodies, including anti-Lyt-2.2 andanti-LFA-1 mAbs, that inhibited antigen-specific lytic activity wereable to promote lysis of the K562 targets by the CTL effectors. Thisability to redirect lysis of the BM10-37 CTL clone to the K562 targethad in fact been used as the screening procedure for identiyfing the145-2C11 mAb. TABLE 1 mAb 145-2C11 Activates Murine T Cells; Evidencedby Induction of Non-antigen-specific Lysis % Specific Lysis Target(K562) Responder Stimulator mAb E/T 100:1 E/T 33:1 E/T 10:1 bm10 B10None 2.8 1.7 1.5 anti-T3-∈ 53.6 42.0 37.0 (145-2C11) anti-Ly-6C 1.0 0.6−0.1 (144-4B11) anti-Thy-1 1.3 −0.1 0.1 (145-7E12) anti-Lyt-2.2 2.2 0.7−0.6 (83-12-5) anti-LFA-1 0.1 0.5 0.5 (H35-89.9) YBR B10 None 3.0 2.12.0 anti-T3-∈ 44.3 34.0 23.5 anti-Ly-6C 3.7 2.9 2.1 anti-Thy-1 3.5 0.81.4 anti-Lyt-2.2 1.9 0.9 1.2 anti-LFA-1 2.0 2.3 2.3

The induction of CTL activity was determined by incubating the effectorCTL and the antigen negative human target K562 in the presence of 5 μlof culture supernatant for 8 hr at 37° C. Similar results were obtainedby using a wide range of antibody concentrations (from 50 μl to 1 μl ofculture supernatant). E/T=effector/target ratio.

In the studies set forth in Table 2, the mitogenic properties of themAbs were assessed by proliferation assays. In particular, the abilityof mAb 145-2C11 to induce T cell proliferation was studied. In thesestudies, T cells (10⁵) were cultured in RPMI-1640 medium containing 10%fetal bovine serum and 50 uM 2-mercaptothanol with irradiated (2000rads; 1 rad=0.01 Gy) spleen cells (2×10⁵) in the presence or absence ofmAb and/or factors in flat-bottomed microwell plates. The recombinanthuman interleukin 2 was provided by Cetus (Palo Alto, Calif.). After 2days, the cells were incubated with 1 uCi (1 Ci=37 GBq) of [³H]thymidineper well for 18 hr, the samples were harvested, and incorporation ofradioactive isotope was measured with a scintillatin counter.

Thus, purified T cells were cultured with 145-2C11 or control mAbs inthe absence or presence of costimulating factors. The 145-2C11 mAbinduced significant proliferation in the absence of exogenous factors.However, the addition of either phorbol 12-myristate-13-acetate orrecombinant interleukin 2 signficantly increased the proliferativeresponse. Finally, both Lyt-2⁺ (CD8), L3T4⁻ (CD4) and Lyt2⁻ (CD8), L3T4⁺(CD4) splenic T cells proliferated in the presence of soluble anti-CD3-εmAb. By comparison, a soluble anti-Vβ8-specific mAb, has been shown tostrongly stimulate CD8 T cells but to only minimally affect CD4lymphocytes. Thus, phenotypically distinct subsets of T cells might bedifferentially activated by antibodies specific for different componentsof the murine TCR-T3 complex. TABLE 2 mAb 145-2C11 Activates T cells asEvidenced by Induction of T-Cell Proliferation [³H]Thymidineincorporated, Responder* cpm ×10⁻³ Exp. Strain Treatment^(t) PMA^(f)rIL-2^(§) Control mAb^(¶) 145-2C11 1 B10 None − − 1.0 61.5 + − 6.2 249.6− + 5.2 258.0 2 B10 C − + 2.6 90.4 CD8 + C − + 3.9 161.6 anti- CD4 + C− + 7.0 57.9 anti- C + − 3.1 137.1 CD8 + C + − 5.2 128.3 anti- CD4 + C +− 6.9 54.3 anti-*C57BL/10 T cells (10⁵) were cultured with irradiated spleen cells (2 ×10⁵) as stimulators.^(t)spleen cells were incubated with antibodies to the CD8 or CD4molecules followed by rabbit complement (C). In both cases, antibodytreatment resulted in the elimination of >95% of the corresponding cellpopulation, as judged by flow cytometry.^(f)Phorbol 12-myristate 13-acetate (10 ng/ml).^(♯)Recombinant interleukin 2 (50 units/ml).^(♭)mAb 145-8D10.

Thus, antibodies against CD3-ε (e.g., 145-2C11) have the ability toprovide immunopotentiation in the forgoing assays, as measured byproliferation, secretion of lymphokines and the expression ofinterleukin 2 receptor (IL-2R). Similar phenomena occur following thetreatment of mice with doses of, e.g., on the order of 4 μg to 400 μganti-CD3 mAb in vivo (30; FIGS. 2-4). At higher doses, however (e.g., onthe order of 40 μg to 400 μg) anti-CD3 mAbs also cause TcR coating,modulation and depletion of T cells from peripheral blood and lymphoidorgans. Thus, the net result of treatment with high doses of anti-CD3mAbs in vivo is immunosuppression. For this reason, the presentinvention is concerned with compositions and protocols which aretailored to allow the administration of relatively lower doses.

It is contemplated that assays such as those set forth above for T cellactivation and potentiation can be employed in a variety of fashions toassist in achieving the goals of the present invention. For example, itis proposed that assays such as these can be employed for screeninghybridoma colonies to identify those which secrete mAbs having thedesired immunopotentiating effect. Similarly, assays such as these canbe readily employed to screen for and identify other suitableimmunopotentiation agents such as immunopotentiating bacterial ormycoplasmal proteins. Furthermore, assays such as these can be employedas an initial step in the determination of appropriate dosages in testanimals (e.g., in terms of mg/kg body weight) which will provideimmunopotentiation as opposed to immunosuppression.

The foregoing information can, in turn, be employed by those of skill inthe art to determine reasonable dosages which would likely be successfulin human therapy. For example, anti-CD3 (OKT3) administered to humansduring a clinical trial on the use of anti-CD3 in cancer treatment, isfound to act in similar fashion to activate T cells as in the above testsystems (FIGS. 5 A and B).

Although immunopotentiating antibodies directed at activation antigensexpressed on all T cells can be used in the practice of the presentinvention, it is proposed that antibodies or reagents specificallydirected at T cell subsets are even more useful as immune adjuvantswithout producing associated immunosuppression, because this specificityensures that the majority of T cells will remain unaffected.

Thus, in the practice of the invention, the potentiation reagents usedwill in certain cases activate all T cells, and in others subsets of Tcells will be selectively activated.

T cell subsets are defined as individual families of T cells that sharecommon features including cell surface proteins such as CD4 and CD8which are expressed on 66% and 33% of the T cells, respectively, Ly-6and CD28 expressed on distinct families of T cells, or the TcR proteins.(11, 15, 16)

Listed below in Table 3 are a series of preferred T cell epitopes whichit is proposed can be employed in the practice of the present inventionto generate immunopotentiating mAbs. TABLE 3 Preferred T CellEpitopes 1. Proteins in the T cell Receptor Complex (TcR) a.Non-polymorphic (Monomorphic) Epitopes (e.g. CD3-γ, δ, ∈, ζ or epitopesfrom the constant region of the TcR α, β, γ, δ chains) b. PolymorphicSubset Specific Epitopes (e.g. epitopes expressed on the variableportion of TcR, Vα, Vβ Vγ or Vδ chains) 2. Proteins Not Associated withthe TcR a. Non-polymorphic (Monomorphic) Epitopes (e.g. Thy-1, CD2) b.Polymorphic Subset Specific Epitopes (e.g. CD4, CDB, Ly-6, CD28)

The response of the immune system to Staphylococcus enterotoxin (SEB) asan immunopotentiating agent is surprisingly stronger than, althoughqualitively similar to, that seen in response to anti-CD3. (FIGS. 7-10).For example, SEB produced a dose responsive increase in allogeneic cellproliferation in mice (FIG. 7). In another assay for immunopotentiation,SEB administration exhibited a surprisingly enhanced IL-2R expression byT cells, and also showed a dose-response. (FIG. 8). Moreover,proliferative effects evoked by SEB administration, as measured in thepresence of RIL-2, were also of a surprising magnitude (FIG. 9).

It is of particular importance to note that, in contrast to anti-CD3,SEB application results in an expansion of a selected subset oflymphocytes, V_(β)8⁺ cells (FIG. 10). V_(β)8⁺ cells are a subset of Tcells representing only 20%, and are important because they mediate avariety of immune responses. Thus, the ability of SEB to preferentiallyexpand V_(β)8⁺ is of particular significance because it activates only asmall subset of polyclonal T cells without directly affecting themajority of the T cell response.

B. Recruitment of Stem Cells and Enhancement of Transplantation

It is proposed that mAb therapy in accordance with certain embodimentsof the present invention will have profound utility in connection withbone marrow transplantation. One of the biggest problems in bone marrowtransplantation is the presence of T cells in the innoculum. Althoughseveral studies have suggested that depletion of T cells is critical inavoiding graft versus host disease (GvHD), T cells may play a criticalrole in engraftment. First, T cells may be required for limitingcytomegalovirus and Epstein-Barr virus infection in transplantedindividuals. Second, when bone marrow transplantation is used inleukemia patients, specific T cells may provide potent graft vs.leukemia responses that eliminate residual tumor. Finally, T cells mayprovide a critical role in engraftment by producing a variety of growthfactors such as GM-CSF. Thus, while total T cell depletion may eliminateGvHD, it may also compromise successful bone marrow engraftment.

It is proposed that aspects of the present invention may be useful inaddressing this problem. There is apparently an interrelation betweenimmunosuppression and immunopotentiation. The use of certain doses ofactivating anti-CD3 antibodies in vivo will not only prevent GvHD butwill also activate endogenous T cells to produce hematopoietic growthfactors that facilitate engraftment. Exemplary doses for achieving thiseffect will range from 5 mg/kg to 20 mg/kg, with about 10 mg/kg beingproposed as optimal. The ability of immunopotentiating agents toincrease hematopoesis is demonstrated, e.g., by studies wherein it isshown that specific recruitment of stem cells such as myelocyte andbands occurs upon administration of such agents in humans, and increasedcolony formation in both mice and humans. In addition, it is proposedthat mAbs directed at activation antigens present on stem cells or otherhematopoietic cells may induce factor production or cell differentiationthat will facilitate engraftment in the absence of T cells.

Importantly, high dose anti-CD3 treatment blocks graft vs. host disease.In an exemplary study, (C57BL/10× BALB/C)_(F1) mice were first treatedi.p. with 250 μg of the anti-CD3 mAb (145-2C11) and then exposed to 500rads gamma-irradiation. Within one to two hours after irradiation, themice were injected with spleen cells from a C57BL/10 mouse. Controlmice, not pretreated with anti-CD3 died of GVH disease within 2 weeks.In contrast, 75% of mice given a single dose of anti-CD3 survived. Notethat the anti-CD3 antibody was preadministered because it is proposedthat circulating anti-CD3 antibody would modulate TcR from host T cellsand thereby inhibit HvD reactions in experiments focused on enhancementof allogeneic bone marrow engraftment. Thus, because circulatinganti-CD3 mAb depleted alloreactive T cells from the donor innoculumbefore GvH reactions can be initiated. (FIG. 13) Additionally, the mAbeffects were examined by CSF assays, in vivo, and CFU-C, BFU-E and CSFassays in vitro.

Studies have demonstrated that mAbs may be directed to recognizeactivation molecules expressed on hematopoietic cells. Severalantibodies have recently been identified that react with subsets ofhematopoietic stem cells. Ly-6A, also known as Scal, is expressed on theearliest hematopoietic stem cells and studies have shown that mAbsdirected at the Ly-6A epitope will activate T cells. A second mAb143-4-2, which reacts with the Ly-6C epitope expressed on 40% ofnon-stem cells in the bone marrow has also been shown to activate Tcells [27]. These results support the belief that mAbs can stimulatebone marrow.

The data in FIG. 12 demonstrates that administration of 1-3 doses of 100μg results in profound increases in circulating stem cells, such asmyelocyte and bands, directly in response to the administration. Asingle dose of 250 μg of anti-CD3 resulted in a >100-fold increase inCFU-C, BFU-E and total bone marrow colonies (FIG. 11) 4 to 10 days postinjection. These increases in hematopoesis have also been observed inhumans treated with OKT3 (FIG. 12). These studies were carried out byhistological examination and standard in vitro bone marrow colonyformation assays in the absence or presence of exogenous growth factorserythropoetin or GM-CSF.

Similarly, Table 4 below presents data demonstrating the in vivo effectsof anti-CD3 on hematopoesis. The studies shown in Table 4 were performedby injecting 250 μg of anti-CD3 on day 0. Bone marrow cells wereharvested on days 4 and 10 and examined in vitro for bone marrow colonyformations as above. As can be seen, the administration of anti-CD3 tomice in all cases resulted in profound increases in hematopoeticprogenitor cell activity. TABLE 4 In vivo effects of anti-CD3 onhematopoiesis Day 1 Day 4 Day 10 NL BM +0 0 0  12.3 +/− 2 +100 U GMCSF  96 +/− 4   98 +/− 2  95.3 +/− 4 +3 U EPO (CFU-C)  7.3 +/− 2  11.5 +/−3  19.3 +/− 1 (BFU-E)  17.8 +/− 3 αCD3-Treated +0  27.3 +/− 3 253.3 +/−5 302.8 +/− 6 BM +100 U GMCSF 238.8 +/− 4   950+/19 416.3 +/− 6 +3 U EPO 56.3 +/− 4 162.5 +/− 3 292.5 +/− 1  15.3 +/− 2 150.8 +/− 3   102 +/− 52. Immunopotentiation Agents as Immunoadjuvants

As noted above, an important aspect of the invention is the recognitionthat immunopotentiation agents described herein can be employed an“immunoadjuvants” in order to evoke an improved immune response tocompounds against which such a response is desired. Thus, it iscontemplated that such immunopotentation agents may be formulated withor otherwise coadministered together with such compounds in order toimprove or to develop an immune response against such compound. Variousstudies have been performed which serve to demonstrate the surprisingpotential for the immunopotentiation agents hereof to act in this manneras immunoadjuvants.

In one such study, the ability of various adjuvant substances werecompared on the basis of their ability to promote the formation ofantibodies against a Keyhole Limpet Hemocyanin (KLH) test antigen. Theadjuvants studied were PBS (phosphate buffered saline control), CFA(complete Freund's adjuvant) and anti-CD3. The study was carried out asfollows: 100 μg of KLH was injected on day 0 i.p. in the presence orabsence of adjuvant. Mice were boosted on day 14 and bled at weeklyintervals. Antibody activity was assessed in sera by standard ELISA.

The results of this study are shown in FIG. 6. As can be seen, at alldilutions of the resultant antiserum, the co-administration of anti-CD3significantly outperformed the PBS adjuvant in terms of the specificanti-KLH titer which developed.

In another series of studies, mice were tested for their ability toproduce specific antibodies in response to challenge by various routeswith a KLH-linked peptide, termed peptide 1005/45-KLH, with and withoutco-administration of anti-CD3. This peptide is derived from theCD4-binding portion of the HIV-GP120 molecule. Mice were administeredthe test immunogen, 1005/45-KLH, either 100 ug subcutaneously (sc) or100 ug intraperitoneally (i.p.) at day 0 and 14, followed byadministration of 4 ug of anti-CD3 (145-2C11) i.p. in test animals. Atvarious time points thereafter (7, 14, 21 and 28 days), the respectivemouse sera was tested for the appearance of antibodies against 1005/45(anti-1005/45 titer). As shown in FIG. 14, the results demonstrated aprofound increase in the 1005/45-specific titer by day 21 in the145-2C11-treated animals as compared to the controls. The strongestresponse, in terms of serum titers of anti-1005/45 antibodies, was inthe group that received both the antigen and 145-2C11 intraperitoneal.(Table 5, FIG. 14) There was essentially no response in the controlgroups or in those treated subcutaneously. The results indicate theimmunopotentiating effect of anti-CD3 as an adjuvant when administeredin conjunction with a relatively non-immunogenic protein. TABLE 5 SERUMTITERS OF ANTI-1005/45 ANTIBODIES IN C57BL/10 TREATED WITHINTRAPERITONEAL (IP) OR SUBCUTANEOUSLY (SC) 1005/45-KLH AND 145-2C11Days post injection Tag# 7 14 21 28 (antigen SC, no 2C11) 1 ND 100 40050 2 ND ND ND ND (antigen SC, 2C11 IP) 3 ND  50 100 50 4 ND 100 50 50(antigen IP, no 2C11) 5 ND ND ND ND 6 ND ND ND ND (antigen IP, 2C11 IP)7 ND 100 800 400 8 ND 200 1600 1600Data are expressed as highest dilution at which antibody was detected inserum by ELISA.ND = not detectable.3. Heteroconjugates

Small peptides have been found to act as immunogens when used withadjuvants other than monoclonal antibodies or bacterial enterotoxins.However, activation of immune system components was not reliable enoughto predict clinical success. Long term, specific immunity is due in manyinstances to the stringent requirements for T cell recognition ofantigens in the context of the polymorphic MHC molecules. The use ofimmunopotentiating proteins, such as monoclonal antibodies and bacterialproteins, to activate T cells in the absence of the requirement forantigen/MHC interaction, coupled with the likelihood that most smallpeptides will express immunogenic epitopes even though they may not berecognized by the T cell or MHC proteins form a major foundation of thisinvention. One object of this invention was to directly couple a peptideto an immunopotentiating protein, e.g., monoclonal antibody or bacterialenterotoxin, to provide a more potent immunogen than a peptide alone.What has been achieved in this invention is to develop a “pass key”which tricks the cell into thinking it sees a foreign antigen beingpresented, causing it to be activated. By presenting a peptide eitherlinked to an immunopotentiating protein, or in combination with it, theT cell activity is directed towards the unique antigen-specific B celltarget. (FIGS. 16 and 17).

For a heteroconjugate, the second protein is isolated and purified bystandard methods from diseased or abnormal tissue, from an infectiousagent, or by genetic engineering a specific amino acid sequence usingstandard molecular biology techniques. Embodiments of second proteins ingeneral are shown in Table 6. A specific embodiment would be the aminoacid sequence, T1, encoded within the gp120 protein of the HIV virus: KQ I I N M W Q E V G K A M Y A. TABLE 6 Embodiments of the secondprotein* in the heteroconjugate Category Size Example Hapten SmallChemical FITC (FIG. 7, 8) Compounds Peptide 8-50aa Peptide 18Protein >50aa gp 120 (HIV Virus)*Includes an epitope against which a humoral immune response is desired.

To complete formation of a heteroconjugate, cross-links are formedbetween the immunopotentiating protein and the second protein by use ofany of the methods which link proteins to form bonds that are stableunder normal physiologic conditions. Such methods include biotin-avidinbridges, SPDP functional groups, and cross-linking of maleimide and SHgroups.

4. Immunotherapy and Immunization

This invention is also directed toward a method of stimulating an immuneresponse in persons identified who are in need of such stimulation dueto having diseases or infections. The heteroconjugate is prepared with apharmaceutically acceptable excipient or diluent to form atherapeutically effective compound. Persons who are candidates for thistreatment include those who have a tumor, are immunocompromised or havecontracted AIDS or other viral, bacterial, fungal, or parasiticinfections.

Another embodiment is to stimulate the immune response by administeringonly the immunopotentiating protein of the heteroconjugate not linked tothe second protein. The immunopotentiating protein may be administeredbefore, after or in conjunction with the second protein. Alternatively,a single immunopotentiating agent may be used.

For clinical use, the persons who are in need of treatment areidentified and the monoclonal antibody which is specific for anon-polymorphic or polymorphic T cell surface protein is administered tothe persons by intravenous route. The monoclonal antibody must not beimmunosuppressive nor adversely affect CD3/TcR expression at the doseused. One embodiment disclosed is to use monoclonal antibody against theCD3 cell surface antigens associated with the TcR. The mAb mimics aphysiologic antigen and bypasses the TcR antigen-specific recognitionmechanism.

If the epitope is a tumor, treatment with doses of monoclonal antibodyselected to be at levels which are not immunosuppressive, butimmunopotentiating, prevents tumor outgrowth and also provides lastingtumor immunity. Methods disclosed in this invention enhance the immunepotential of T-cells and other components of the immune system andprovide powerful tools to amplify the immune system. An object of theinvention is to augment the effects on the immune system of weaklyimmunogenic small peptides or proteins. For example, conjugatingproteins to monoclonal antibodies produces a stronger response bybypassing the requirements of immunogenicity mandated by MHC restrictionand antigen recognition by T-cell receptor.

The strategy of this invention is to stimulate T cell immune reactivityusing mAbs. T cell activation enhances the ability of an individual toreject a malignant tumor in a specific manner and produces long lastingtumor immunity. Therefore, the antigen-like effect of monoclonalantibodies to TcR/CD3 structures are disclosed as a means tospecifically enhance immune function in vivo in tumor bearingindividuals.

This invention allows for the direct interaction of the vital componentsof the immune response in developing strong cellular and humoralimmunity. It is directed also toward protecting those who are not yetaffected with the disease or condition by disclosing a vaccinecomprising the immunogenic heteroconjugate described above. The use ofthis heteroconjugate does not require the interaction of the peptidewith the major histocompatibility complex antigens, which presents avery significant advantage, that is, bypassing the polymorphic responsewhich is usually a problem with the MHC system. All immune responses aredependent on the ability of T cells to recognize processed antigenassociated with major histocompatibility antigens (MHC).

Any vaccine approach which utilizes HIV peptides or inactivated virusantigen must depend on the ability of antigenic peptides to bind theappropriate MHC antigens necessary to initiate an immune response.Specifically, the tremendous polymorphism of the MHC antigens expressedin the population and the variation of the virus, developing asuccessful HIV vaccine for general use faces major obstacles. Thus,developing an ideal form of immunotherapy and vaccines against, forexample, AIDS, could be achieved by increasing the antigenicity of thepeptide MHC interaction or boosting the activity of the immune cells,thus reducing the threshold of antigenicity necessary to triggerspecific immune responses and memory following antigen exposure. A moregeneral vaccine is created by use of the heteroconjugate which is anaspect of this invention because the extensive genetic diversity of theMHC is not a factor in binding to T cells. Given the highly polymorphicnature of both the MHC proteins and the TcR gene products, the number ofpeptides that are present in sufficient quantity to bind both the MHCand TcR, thus inducing an immune response, is small, factors limitingthe success of vaccines.

The efficacy of vaccines, in particular those which may be weaklyimmunogenic, may be improved by modifying the foreign antigen such thatit is more immunogenic, or allowing the use of peptides which are notimmunogenic under normal conditions because they would not bind MHC, butwhich may constitute a conserved site on the major HIV viral proteinsthat is found in a large percentage of the population. In oneembodiment, vaccines are comprised of heteroconjugates. Monoclonalantibodies which are used as the immunopotentiating protein in thevaccine include T-cell surface antibodies directed againstnon-polymorphic or polymorphic T-cell surface molecules. The secondprotein which is used as the other portion of the heteroconjugateincludes those which are not capable of stimulating the immune systemsufficiently to achieve immunization unless they are administered in aconjugate with the monoclonal antibody.

An object of this invention is an approach to therapy which activatesthe host antitumor cellular effector mechanisms, even though the epitopeis only weakly immunogenic and could evade host recognition andrejection unless augmented. Major forms of immunodeficiency may be dueto the inability of antigen to trigger a primary immune response due tosuboptimal antigen presentation and T cell activation.

In addition to the administration of compositions for stimulatingimmunity, the present invention also contemplates that certainimmunological products which are produced as the result of such anadministration may provide a benefit to some individuals, particularlyimmunocompromised individuals. That is, it is contemplated that theimmunopotentiating compositions of the present invention may be employedto produce antibody compositions, such as gamma globulin fractions,which may be administered to individuals for the development of passiveimmunity.

For such embodiments, immunopotentiating compositions hereof whichcontain appropriate compounds against which an immune response isdesired, whether such compositions are in the form of “adjuvant”compositions or heteroconjugates, are administered to disease-freeindividuals in an amount effective to elicit a specific immune responseagainst the second compound. The resultant gamma globulin fraction isthen obtained and purified by well known techniques, and administered ineffective amounts to individuals in need of such treatment. Note that inaddition to the use of immunoglobulin fractions, the present inventioncontemplates that other immunological products, such as activated T andB cells will also be useful for such purposes, e.g., in the treatment ofcancers and even HIV infections.

5. Summary

The development of the invention required production of theimmunopotentiating agents, testing their effects in vitro, and finallydeveloping their in vivo effects in mice as models, and in humans forpurposes of treatment.

The in vivo effects disclosed in this invention are not absolutelypredictable from the in vitro effects because an intact organism'sresponse reflects interactions of a complex immune system, whereas, invitro, individual components can be controlled, and system interactionsare difficult to simulate. In vivo, various components which reactedseparately in the laboratory in certain fashions, may not interactappropriately when their actions are combined.

Methods

The following examples present methods for preparing theimmunopotentiating agents described herein, methods for administeringthem to non-humans or humans, and methods for assaying their effects.The techniques employed in the following examples reflect those found orcontemplated by the present inventor to constitute preferred modes forthe practice of the invention. However, those of skill in the art willappreciate in light of the present disclosure that many modificationsand changes may be made in the disclosed techniques without departingfrom the spirit and scope of the invention.

EXAMPLE 1 Preparation of the Immunopotentiating Agents

1. Single Agents

A. Monoclonal Antibodies

Monoclonal antibodies were prepared against specific classes of T cellepitopes. These classes are listed in Table 3. In the followingembodiment, methods for preparing a monoclonal antibody against anonpolymorphic epitope, CD3, and polymorphic epitope Vβx where x=aspecific chain in the variable part of the TcR complex, are described:

(1) Preparation of a mAb Directed Against the Murine CD-3 Chain of theTcR Complex

Monoclonal antibodies were generated which are reactive with the T cellsurface structures expressed as alloreactive cytotoxic T cell (CTL)clones and involved in T cell activation. MAbs specific for these cellsurface molecules were identified using an assay (developed byBluestone, the redirected lysis assay (33), see also Example 13) basedon the ability of antibodies reactive with the TcR complex to induceantigen-specific CTL to lyse cells which are not their natural targets.This activity depends on the ability of the antibody to both activatethe CTL via the TcR and multivalently crosslink the effector to thetarget cells via the Fc receptor (FcR) on the target cells. Several mAbshave been derived in this fashion. This assay is one way to select formAbs that activate T cells, and increase proliferation and lymphokinerelease.

The mAb, 145-2C11, was derived by fusing spleen cells from an Armenianhamster immunized with a murine CTL clone to SP2/0 cells, and screeningthe resulting hybridoma supernatants in the redirected lysis assay. ThismAb immunoprecipitated the complete TcR complex using non-ionicdetergents but reacted specifically with a 25-kD protein component(CD3-ε) of the antigen-specific TcR complex. 145-2C11 mAb may also beobtained by growing hybridoma cells in an Acusyst P machine(Endotronics, Minn., MN) and then collecting the supernatant. Antibodiesare then purified by 50% ammonium sulfate precipitation followed by gelfiltration on an ACA 34 Ultragel column [BF Biotechnics, Savage, MR]

Another mAb, 143-4-2, was derived by fusing spleen cells from a BALB/cmouse immunized with a murine CTL clone and screened as in Example 13.This mAb defines a novel cell surface molecule involved in T cellactivation. The expression of the 143-4-2 defined epitope was expressedon the Ly-6C molecule and was restricted in its lymphoid expression tobone marrow cells and to a subset of peripheral CD8+ cells (34). Theanti-Ly-6.2C antibody can promote the lysis of target cells that do notbear antigens by alloreactive CTL clones and, in the presence ofcofactors (PMA or IL-2), induced a subset of CD8+ cells to proliferate,perhaps through an autocrine pathway. Although the antibody describedhas antigen-like effects as described for anti-TcR complex reagents, itwas shown that the Ly-6.2C molecule was not associated on the cellsurface with components of the TcR complex. These included biochemicalanalyses, phenotypic studies and functional studies that showed that,unlike activation via the TcR/CD3 complex, Ly-6.2C-mediated activationwas not inhibited by anti-CD8 mAbs (35). Nevertheless, cell surfaceexpression of the TcR complex is required for optimal triggering of Tcells via the Ly-6.2C molecule.

mAbs were purified by ion exchange chromatography and gel filtration.F(ab′)2 and Fab fragments were prepared by pepsin and papain cleavage ofpurified antibody, respectively (36).

Additional mAbs were prepared that can be employed as immunopotentiatingproteins in vivo to activate T cell subsets and hematopoietic stemcells. These include: UC3-10A6 and UC7-13D5 mAbs which are specific forthe TcR δ receptor; UC3-7B7 which is specific for Thy-1; 145-4B11 and D7(obtained from Tom Malek, Miami, Fla.) which are specific for Ly-6A(also known as the Scal antigen expressed on bone marrow stem cells);and H597.57, an anti-TcRαβ mAb (Ralph Kubo, Denver, Colo.).

(2) Preparation of a mAb Directed Against Vβx, Wherein x is a SpecificChain, e.g., Vβ2

Immunopotentiating proteins disclosed in this invention comprisemonoclonal antibodies prepared as follows:

-   -   a. expand lymphocyte cell cultures by standard methods (37); use        limiting dilution or hybridoma technology to clone T cells or        produce T cell hybridoma (38) thereby being able to select        discrete activation of cells from a single progenitor cell        (clone);    -   b. use molecular probes or mAb to determine Vβx usage; this will        identify all clones which express Vβx, (although these clones        will also express other T cell antigens including a Vα chain,        CD3 chains, and non-TcR associated proteins);    -   c. use clones or extracts of the clones expressing Vβx to        immunize animals (mouse, rat or hamster), by standard techniques        (39);    -   d. using spleens from the immunized animals, prepare hybridomas        by standard techniques (40)    -   e. screen the hybridomas for those which satisfy both of the        following criteria:        -   1) activate T cells (determined by assays described in            Example 13) (Note: of 1000-2000, mAb screened, a minority            will activate T cells)        -   2) produce a monoclonal antibody directed to the T cell            clone;    -   f. select monoclonal antibodies which are specific only for Vβx,        by eliminating mAb which activate clones which express with        other Vβ proteins, based on the reactivity pattern of different        Vβ-expressing clones, and biochemical analysis of TcR usage.    -   g. confirm that the antibody is specific for Vβx by biochemical        techniques including immunoprecipitation and Western blots of        the TcR antigen or Vβx and other T cell clones.

B. Preparation of an Immunopotentiating Agent Wherein this Agent is aMicrobial Protein which Activates T Cells

Any microbial component, e.g. a bacterial enterotoxin, may be assayedfor T cell activation. Enterotoxins may by purchased from biochemicalsuppliers. Toxins that satisfy criteria of activation assays may besubjected to standard protein purification techniques, gel filtrationand exchange chromatography. (1) At each step of the purification, theresulting product is assayed to determine whether the ability toactivate T cells is preserved. Preferred epitopes may be selected fromthose shown in Table 3.

EXAMPLE 2 Activation of T-cells by Administration of Anti-CD3

To evaluate whether low doses of anti-CD3 were effective as activating Tcells in mice, mice were given different doses and their lymph nodes andspleen cells were examined for IL-2R expression by flow-cytometry. IL-2Rexpression was enhanced at the three doses tested (4, 40 and 400micrograms) and plateaued at 400 micrograms. (FIG. 2). When the samelymphoid cells were incubated in media containing human rIL2, theirproliferation was enhanced in proportion to their IL-2R expression. Theimmune suppression which results from a dose of 400 microgram ofanti-CD3 was the result of T cell depletion, T cell receptor blockadeand modulation of the TcR complex. The net result was that the amount ofcell surface CD3 available to react with antigen was decreased,rendering the T cell unable to respond to antigenic stimuli becauseefficient antigenic specific activation depends on the presence ofintact TcR. Therefore the quantity of available CD3 on lymph node cellsfrom control and treated mice were examined 18 hours after treatment.Cells were stained with fluorescein isothiocyonate (FITC)-conjugatedanti-CD3 and Thy-1+ T cells and were examined. (FIG. 2). Optimalconditions for anti-CD3 mediated T cell activation are those in whichthe concentration was 4 μg, low enough to permit activation withoutadversely affecting CD3/TcR expression. Additional evidence that the lowdose anti-CD3 treatment not only nonspecifically activated T cells butincreased immune responsiveness was obtained from allogenic mixedlymphocytes reaction (MLR) and mixed lymphocytes tumor culture (MLTC)studies.

EXAMPLE 3 Effect of Anti-CD3-Treatment on Sendai Virus Infection in Mice

The purpose of this example was to test the effect of mAb treatment oninfection. Administration of low doses of anti-CD3 prevented the lethalpneumonia caused by the Sendai virus in >60% of mice. Anti-CD3 treated,virally-infected mice also developed lasting virus-specific immunity asevidenced by their ability to withstand a subsequent dose of Sendaivirus of 1000 times the LD₅₀ dose. Treated mice also developed a Sendaivirus specific DTH and antibody response similar to mice immunized witha non-virulent Sendai virus vaccine. Interestingly, the 129/J strain ofmice were also protected by the anti-CD3 treatment. Because virussusceptibility in these mice has been shown to be caused by aninadequate generation of NK cell responses, it appears as if NK activityinduced by the mAb treatment contributes to viral protection. (51)

EXAMPLE 4 Anti-CD3 Prevents Malignant Progressor Tumor Growth in Mice

The purpose of this example was to determine the feasibility ofimmunotherapy designed to prevent tumor outgrowth and induce tumorimmunity by administering anti-CD3 in vivo to activate T cells. Thispossibility was tested in mice with the eventual objective of pursuingsimilar strategies in humans.

Effects of the anti-CD3 treatment on malignant tumor growth in vivo weretested in mice. The C3H fibrosarcoma 1591-Pro-4L a weakly immunogenicultraviolet-light-induced murine tumor that lacks cell surface CD3 andFcR does not react directly with the anti CD3 used for treatment. Thismalignant tumor grows progressively in 95 percent of normal CD3H miceand eventually kills the mice by infiltrative growth without macroscopicevidence of metastases. None of the mice treated with 4 μg of anti CD3developed tumors in this experiment. (Table 7). Animals treated with 4μg of anti CD3 also developed tumor immunity because they failed todevelop tumors following a second inoculation of Pro-4L 60 days later,despite no additional intervening monoclonal antibody therapy, whereascontrol animals challenged with tumor fragments at that time developedtumors. Treatment with F(ab′)₂ is immunosuppressive but does notactivate T cells and had no effect on tumor growth.

Immunopotentiating effects include increased tumor specific T cells,lymphokine-activated killer cell activity, increases in tumor necrosisfactor (TNF) detected in the serum of treated, not control mice.Findings reported in Table 7 were confirmed using a metastatic tumorsystem MCA102. Lung metastases in mice having established MCA102 tumorsare significantly reduced by the mAb treatment. In one experiment, miceinjected with tumor alone developed a mean of 105±26 lung metastaseswhile mice treated with anti-CD3 mAb 3 or 10 days after tumorinoculation developed 25±6 and 44±14 lung metastases respectively. TABLE7 Summary of Tumor Incidence at Day 28 in Anti-CD3-Treated Mice Therewas no recurrence, late outgrowth, or tumor regression after 28 days.The P value (X² method) for the difference between the control animalsand those treated with 4 μg of anti-CD3 was P < 0.001. Tumor incidence(day 28) Treatment group no. with tumor/ (three separate experiments)total no. (%). Control 42/44 (95) Anti-CD3 (4 μg) 11/31 (35) F(ab′)₂anti-CD3 (2.6 μg)  9/10 (90)*P < 0.001

EXAMPLE 5 The Use of Staphylococcal Enterotoxins (SE) asImmunopotentiation Agents

The recent demonstration that Staphylococcal enterotoxins (SE)specifically activate T cells which express certain VP subsets (55)provide an approach for in vivo T cell activation in accordance with thepresent invention. These reagents, which activate in vivo have severaladvantages: 1) The SEs activate more than one Vβ-subset in vivo; 2) TheSEs cause minimal TcR modulation and immunosuppression at low doses; 3)The SEs induce T cells to proliferate in vivo; and 4) Results using theSEs can be directly related to man since these reagents react with humanT cell subsets in a similar manner (56).

Results of studies with SEB are presented in Tables 8a and 8b below. Asshown in Table 8a, in vivo administration of SEB results in a selectiveactivation of Vβ8⁺ T cells. Moreover, the studies shown in Table 8bdemonstrate that while two out of six mice treated with four μg ofanti-CD3 developed tumors following tumor innoculation, theadministration of 50 μg of SEB resulted in no incidence of tumordevelopment. TABLE 8a IL-2 Receptor Expression on T Cells from SEBTreated Mice. % CD3⁺ % CD3⁺ % Vβ8⁺ % Vβ8⁺ SPLEEN/LYMPH NODE IL-2R⁺IL-2R⁻ IL-2R⁺ IL-2R⁻ CONTROL SPLEEN 1 25 0 5 ANTI-CD3 (4 μg, 18 HR) 20 76 0 SEB (50 μg, 18 HR) 4.5 17 4 0 CONTROL LN 5 65 2 18 ANTI-CD3 (4 μg, 6DAYS) 4 64 2 21 SEB (5 μg, 6 DAYS) 4 64 2 37

TABLE 8b Tumor regression in anti-CD3 and SEB treated mice. Group TumorIncidence (Day 28) CONTROL 13/16 ANTI-CD3 (4 μg)  6/20 SEB (50 μg)  5/21p < 0.01

EXAMPLE 6 Alloresponse and Anti-tumor Response to SEB

To test the proposed use of SEB for immunopotentiation, C3H mice weretreated either with anti-CD3 (145-2C11) or with one of three doses ofStaphylococcus enterotoxin (5 μg, 50 μg, 500 μg). There were untreatedcontrols. Both syngeneic and allogeneic responses were determined by ³Huptake of lymphocytes. As shown in FIG. 7 treatment with this singleagent promoted an allogeneic response. A clear dose effect was alsoobserved.

The surprising proliferative response of mouse lymphocytes to SEB alone,compared to anti-CD3 is shown in FIGS. 7-10, indicating that theseenterotoxins are useful for immunotherapy. In fact, mice treated withSEB showed a significantly lower tumor incidence (Table 8b).

A difference was found between responses of the immune system to asingle agent in the embodiment of anti-CD3 versus SEB. Anti-CD3 appearedto induce a more generalized response, whereas SEB could be directed toactivate specific T-cell subsets. FIG. 8 presents results of SEBtreatment of mice in vivo, in which IL-2R expression showed a clear doseresponse effect. Specific and preferential stimulation and expansion ofVβ8+ cells in the SEB-treated mice is further shown in FIG. 10.

EXAMPLE 7 Response of Human Lymphocytes In Vivo to OKT3

Confirming the anti-CD3 responses in animal models, FIGS. 5A and B, 11,12 illustrate the response of human cells to low doses of OKT3.Different lymphocyte subsets show a similar pattern of response, butdifferent absolute percentages of total lymphocytes. These results arefrom the ongoing clinical trial of OKT3 in treating patients withcancer. The methods for this trial are described in Example 12.

EXAMPLE 8 Anti-CD3 Treatment Abrogates Graft Versus Host Disease (GVHD)

The use of anti-CD3 will depend on the clinical condition. Depending onthis, doses are selected. Mice were treated with 500 R radiation tosuppress their immune response thereby facilitating transplantation.After 13 days, however, the graft was rejected in control animals.Dramatic increased survival of 75% was observed at the same point intime for animals treated with 250 μg of anti-CD3, and 30% survival after25 μg of anti-CD3. (FIG. 13). This effect is believed due to inducedhematopoesis which revitalizes the graft.

EXAMPLE 9 Use of a Hapten or Peptide as a Second Protein in aHeteroconjugate or as a Combination Administered Concurrently

Fluorescein isothiocyanate (Sigma) or Dinitrophenol (DNP) in boratebuffer, pH 8.5 for 4 hours at 22° C. are examples of haptens tested forthis purpose.

Examples of other second proteins are synthetic peptides of HIV, 13-23residues long prepared by the multiple simultaneous peptide method ofsolid-phase synthesis in polypropylene mesh. These are coupled to theanti-CD3, BSA OVA or KLH using Maleimide as described in Example 2C.vSC-8 (recombinant Vaccinia vector containing the bacterial LACz gene),and vSC-25 (recombinant Vaccinia vector expressing the HIV envglycoprotein gp160 of the HTLV III_(b) isolate of HIV structural orregulatory proteins) is used. Mice are conventionally immunized with 100μg HIV/pep-KLH, or HIV/pep in Complete Freund's Adjuvant (DifcoLaboratories) intraperitoneally 1-12 months before use eitherconcomitantly or after injection of 4 μg of anti-CD3 mAb. Alternatively,mice may be immunized with anti-CD3 coupled FITC or HIV peptide.

Although concomitant treatment of mice with anti-CD3 mAb augmentedantibody production specific for the FITC hapten, the most profoundeffect occurred when the anti-CD3 was directly coupled to FITC (FIG.16). Anti-CD3-coupled TNP, FITC, and Ovalbumin (OVA) peptides arealternative immunogens. These studies provided insights into the abilityof anti-CD3 to augment T cell and B cell immunity. They also providedthe basis for an analysis of the ability of anti-CD3/haptenheteroconjugates to induce immunity by direct T cell/B cell interactionsthat may alter MHC restriction and IR gene control. These studies arebest performed using in vitro plaque forming cell (PFC) responses orELISA to examine the immunogenetics of antibody production. Forinstance, utilizing classic MHC restriction analyses to study the roleof MHC/peptide interactions in anti-CD3 augments immunity, studies haveshown that certain mice are low responders to OVA, depending on theirMHC haplotype. The low response was due to the inability ofcarrier-specific T cells to recognize OVA peptides in the context ofself MHC.

Anti-CD3 is coupled to various OVA peptides including amino acids323-334 which is known to be immunogenic in H2^(d) strains as well asother peptides not capable of generating an immune response (46). Theseexperiments were performed to assay the ability of anti-CD3/haptenheteroconjugates to induce immune responses in low responder haplotypes.In particular, the activation of B cells by a direct crosslinking of Tand B cell by the heteroconjugate is believed to bypass the requirementfor antigen presentation by MHC molecules (FIG. 17). Thus, these modelantigens provides the basis for initial analyses of the immuneregulation in anti-CD3-treated mice.

EXAMPLE 10 Bispecific Ligands

Studies have also been conducted wherein the immunopotentiating effectof employing two monoclonal antibodies linked together to form a singlemolecule. The antibodies employed in these studies were directed againsttwo distinct but specific epitopes, CD3 and CD4. These epitopes wereselected because the anti-CD3 activates T cells, whereas the anti-CD4focusses the activation towards a specific subset. This conjugate wasconstructed by crosslinking the respective antibodies using SPD asdescribed in Example 15B hereinbelow.

In the studies which form the basis of the present example, 4 μg of theresultant conjugate, designated F(ab′)₂ anti-CD3 x anti-CD4, wasadministered to mice i.p. on day 0, and the resultant % of IL-2receptor⁺ cells determined by flow cytometry on spleen cells 18 hourspost injection. The % of IL-2 receptor⁺ cells is an indicator of T cellactivation. The results of these studies are shown in FIG. 15.

As can be seen, the results shown in FIG. 15 demonstrate that thisbispecific antibody construct selectively activates CD4⁺ cells. Thus,the bispecific antibody conjugate is very highly active in terms ofimmunopotentiation.

Materials EXAMPLE 11 Tumors

The UV-induced skin tumor lines 1591-Pro-4-L, 6139-Pro-1, 1316-Pro-1 and1591-Var4L were produced as described (50). Tumor cells were passed byserial culture in complete media. For in vivo studies, tumor cells wereinjected into C3H nude mice. Solid tumors isolated 3 weeks later werecut into 2 mm fragments, and 10 of these fragments were injected into asingle subcutaneous location using a 14 gauge, 2 inch long trocharneedle. Tumor presence and size were determined at one month in mice.YAC-1 T lymphoma cells and P815 mastocytoma cells were used as targetsin some assays in vitro.

Specific antigens may be prepared from tumor cells isolated byserological techniques or suspension dilutions. These antigens werepurified by standard methods, for example, density gradientcentrifugation or velocity immuno-sedimentation. The identity of theantigen was confirmed by serology or histological examination. Cells arelysed and used in the assays described in Example 3 to identify uniquetumor antigens. Protein extracts are prepared by standard methodsincluding molecular sizing and ion exchange chromatography. Furthermolecular and biochemical analysis for T specific antigens may beperformed by established cloning techniques.

EXAMPLE 12 Animals

C57BL/10 male mice between 8 to 12 weeks of age were obtained from TheJackson Laboratory, Bar Harbor, Me. A thymic BALB/c mice and NIH Swissmice were obtained from the National Institutes of Health small animalproduction facility.

EXAMPLE 13 Administration of the Heteroconjugate or ImmunopotentiatingAntibody to Patients

In vivo response to OKT3 is shown in FIGS. 5 A and B, 11, 12. Patientsselected for treatment were those who have histologically documentedmalignancy which is either evaluable or measurable and for whichstandard therapy is unavailable. These patients should not have hadprior administration of murine antibodies or history of documented orsuspected life threatening immune mediated disorders such as asthma.There should not be concurrent drug therapy or infection. Patientsshould not be pregnant or have either class III or IV cardiac disease.(New York Heart Association Classification) The monoclonal antibody tobe administered in a preferred embodiment was Muromonab-CD3 (OKT3), amurine monoclonal antibody directed against the T3 (CD3) antigen ofhuman T cells. This antibody reacts with a 20,000 dalton molecule (CD3)in the membrane of human T cells that has been associated in vitro withthe antigen recognition structure of T cells, and which is essential forsignal transduction. In the in vitro cytolytic assays, low doses of OKT3enhances both the generation and function of effector cells. In vivo,anti-CD3 reacts with most peripheral blood T cells and T cells in bodytissues but has not been found to react with other hematopoieticelements or other body tissues. It is a potent mitogen in vivo.

The drug was administered through a needle in the contralateral vein byintravenous infusion. Moriomurab-CD3 was pushed over 30 to 60 secondsusing a 20 cc syringe containing OKT3 dose in 5% human serum albumin.The patient's vital signs were monitored every five minutes for thefirst fifteen minutes, then at two hours and four hours untiltwenty-four hour post infusion. Patients received 3 treatments at 2 weekintervals unless precluded by toxicity. Toxic response is defined bypublished criteria.

Orthoclone OKT3 (Muromonab-CD3) sterile solution is a murine monoclonalantibody to the T3 (CD3) antigen of human T cells which functions as animmunosuppressant. The antibody is a biochemically purified IgG2immunoglobulin with a heavy chain of approximately 50,000 daltons and alight chain of approximately 25,000 daltons. It is directed to aglycoprotein with a molecular weight of 20,000 in the human T cellsurface which is essential for T cell function. Because it is amonoclonal antibody preparation, Orthoclone OKT3 sterile solution is ahomogenous reproducible antibody product with consistent measurablereactivity to human T cells. Each 5 ml ampule of Orthoclone OKT3 sterilesolution contains 5 ml (1 milligram per ml) of Muromonab-CD3 in a clearcolorless solution which may contain a few fine translucent proteinparticles. Each ampule contains a buffer solution (Ph 7.0±0.5) ofmonobasic sodium phosphate (2.5 ml), sodium sulphate (9.0 mg), sodiumchloride (43 mg), and polysorbate 80 (11.0 mg) in water for injection.The proper name Monomurab CD3 is derived from the descriptive “murinemonoclonal antibody.” The CD3 designation identifies the specificity ofthe antibody as the cell differentiation (CD) cluster 3 defined by theInternational Workshop on Leukocyte Differentiation Antigens.

Orthoclone OKT3 is supplied as a sterile solution and packed in 5 mlampules containing 5 ml of Muromonab-CD3. These vials are stored,refrigerated at 2-8° C. Prior to administration the Orthoclone OKT3protein solution may develop a few fine translucent particles. Thesehave been shown not to affect its potency. The product is prepared forinjection by drawing the solution from the vial into a syringe through alow protein binding 0.2 micrometer filter. The filter is discarded andthe needle attached for IV bolus injection. Orthoclone OKT3 is deliveredas an IV bolus in less than one minute. This drug should not beadministered as an intravenous infusion or in conjunction with otherdrug solutions. When necessary, Orthoclone OKT3 solution may be dilutedwith sterile saline.

For the preparation of OKT3 for injection, the antibody was drawn into asyringe as described above. The appropriate dose of OKT3 was then addedto 15 to 20 cc 5% human serum albumin in normal saline and placed in a20 cc syringe. Toxicity was monitored and may include fever and chills,shortness of breath, allergic reaction, chest pains, vomiting, wheezing,nausea, diarrhea or tremors.

Measurement of the effect of CD3 was performed as shown in Table 9.TABLE 9 Measurement of the Effect of CD3 Treatment on the Immune SystemI. Peripheral Leukocyte Phenotype Total Lymphocyte Count. Total T-CellCount. Total Th and Ts counts. Helper/suppressor Total NK Cell count.CD3 and CD25 Expression. II. Lymphokine Production Interleukin-2 (IL-2)Level. Soluble IL-2 receptor - a reflection of T-Cell Activation. G-CSFand GM-CSF Levels. TNF Level. III. Cellular Cytotoxicity. NK CellCytotoxicity on K562 Cell Line at 0 and 24 hours. LAK Cell Cytotoxicityon Raji Cell Line at 0 and 24 hours. Pre-LAK Cell (LAK Cell CytotoxicityAfter 3-day Culture in IL-2) IV. Cellular Proliferative Responses.Anti-CD3 Induced proliferation. IL-2 Induced Proliferation.Unidirectional MLC against pooled, Mitomycin C- Treated Lymphocytes.PHA- and Con A-induced proliferation.LAK = Lymphokine activated killer cells

Assays EXAMPLE 14 Assays for T Cell Activation

A. Cytotoxicity Assays

Effector cells and ⁵¹Cr-labeled target cells were added to wells of96-well tissue culture Seroclusters (Costar, Cambridge, Mass.) atvarious effector to target ratios, in triplicate, in a total volume of200 μl of complete medium. The monoclonal anti-TcR antibody, 2.4G2 (47),was added to wells at a concentration of 10% cs where indicated. Plateswere then incubated for 4 or 6 hours at 37° C. following which 100 μl ofsupernatant was aspirated and analyzed on a gamma counter (MicromedicSystems, Inc., Horsham, Pa.). Percent specific lysis was calculated byusing the formula [E^(cpm)−S^(cpm)/T^(cpm)−S^(cpm)]×100. E^(cpm) whichrepresents the ⁵¹Cr released from the target cells incubated witheffector cells; T^(cpm) represents release of radiolabel from targetcells in a 0.05N solution of HCL; S^(cpm) represents background releaseof target cells cultured in media in the absence of effector cells. Ingeneral, the data should represent at least three experiments withidentical results. Standard errors for all ⁵¹Cr release values should beless than 5%.

B. Cell-Mediated Lympholysis ⁵¹Cr-Release Assays

Virus-specific CTL populations were generated in vitro using 5×10⁶splenic responder cells mixed with 2.5×10⁶ irradiated (3300 rad)Vaccinia virus infected syngeneic spleen cells (1 hr., 37° C.,multiplicity of injection, 10:1) in 2 ml cultures in complete media(RPMI 1640 supplemented with 10% selected fetal, calf serum (FCS),sodium pyruvate, nonessential amino acids, glutamine, and2-mercaptoethanol). Cytolytic activity is assayed after 6 days onappropriate virally-infected targets or peptide-pulsed syngeneic orallogeneic ⁵¹Cr-labeled targets.

C. Proliferation and Lymphokine Assays

T cells were cultured in RPMI 1640 medium containing 10% FCS and2-mercaptoethanol, in the presence or absence of mAbs and/or factors inflat-bottomed microliter 96 wells plates. In some experiments purifiedanti-CD3 mAb were immobilized on plates as a means of activating cellsin the absence of Fc receptor⁺ cells. After three days, the cells werepulsed with 1 μCi [3H] thymidine for 16 hours, the samples wereharvested and incorporation of radioactive isotope measured using ascintillation counter. The secretion of soluble lymphokines weremonitored by the ability of culture supernatants of activated T cells tosupport the growth of the lymphokine-dependent HT-2 cell line. 5×10³HT-2 cells were cultured for 24-36 hours with the supernatant to beassayed, then pulsed for 16 hours with 1 μCi [3H] thymidine.

This protocol is adaptable in time and cell number when assayed inhumans versus animals.

D. Assays of In Vitro Antibody Response.

Antibody responses were assayed in vitro by two methods: (1) Plaqueforming cells (PFC) responses were measured on the day of culture byassaying TNP, HIV peptide of FITC-conjugated sheep erythrocytes asdescribed (48). Indirect IgG responses were evaluated by blocking theIgM plaques with goat-anti-mouse IgM and developing with a rabbitanti-mouse IgG. (2) Direct binding enzyme-linked immunosorbant assays(ELISA) of FITC, TNP or peptide binding antibodies were carried out asfollows. Nunc ELISA plates were coated overnight with 100 μl of 50 μg/mlFITC-BSA, TNP-BSA or HIV-peptide-BSA in Voller's buffer (4° C.) Plateswere washed extensively with 0.05% Tween 20 diluted in PBS. Free siteswere blocked by incubating the plates with 0.5% BSA for 1 hr. at roomtemperature. One hundred μl of control or immune sera, diluted inPBS-Tween was added to each well in 3-fold serial dilutions andincubated for 1 hr. After washing, 100 μl of 1 μg/well horseradishperoxidase conjugated goat anti-mouse IgG were added. After 1 hr, plateswere washed and 100 μl of peroxidase substrate system were added. Thisreaction was developed at room temperature for approximately 20 minutes,stopped with 100 μl of 1% SDS, and read at 415 nm with an automaticELISA plate reader. ELISA assays was performed by titrating sera, andthe optical density (O.D.) values reported will be obtained from alinear portion of the titration curves where O.D. is proportional tosera concentration. Where appropriate, affinity-purified mouseanti-FITC, anti-DNP or HIV antibodies will be prepared, and experimentalresponses was determined as μg/ml of antibody based on this standard.

E. Specific Embodiment of Assays for Humans Treated with OKT3

I. Control Protocol

-   -   1. Make at least six replicates for control cells.    -   2. Plate 100,000 cells/well in a total volume of 200 uL.    -   3. Incubate at 37 deg C. and 5% carbon dioxide for same amount        of time as stimulated cells.    -   4. Add 20 uL of MTT (5 mG/mL in PBS).    -   5. Incubate for 5 hours at 37 deg C. and 5% carbon dioxide.    -   6. Centrifuge for 5 minutes at 600×g.    -   7. Aspirate 160 uL supernatant carefully.    -   8. Add 160 uL DMSO to each well.    -   9. Agitate plate for 30 minutes or until all crystals dissolve        completely.    -   10. Read immediately on Multiskan at 550 nm.        II. Patient Sample Protocol    -   1. Make at least six replicates per stimulant.    -   2. Plate 100,000 cells/well in a total volume of 180 uL.    -   3. Add stimulant in 20 uL volume to well.    -   4. Incubate at 37 degrees C. and 5% carbon dioxide.    -   5. Add 20 uL of MTT (5 mg/mL in PBS).    -   6. Incubate 5 hours at 37 deg C. and 5% carbon dioxide.    -   7. Centrifuge at 600×g for 5 minutes.    -   8. Aspirate 160 uL supernatant carefully.    -   9. Add 160 uL DMSO to each well.    -   10. Agitate plate for 30 minutes or until all crystals dissolve        completely.    -   11. Read immediately on Multiskan at 550 nm.        Pre-Treatment and 24 Hour Time Points:

Put cells in culture as per proliferation assay and triplicate plateswith LPS (U plates).

After 18-24 hours, remove 150 uL of cell suspension and freezesuspension and pellets at 70°.

Assay pellets for tissue factor.

Assay suspension for TNF-α and IL-1β

EXAMPLE 15 Biochemical Analysis

Cell surface-labeling with ¹²⁵I (Amersham Corp., Arlington Heights,Ill.) was performed by the lactoperoxidase method as describedpreviously (52). Labeled cells were lysed in 1% digitonin buffer, andprecleared with hyperimmune rabbit serum. Lysates wereimmuno-precipitated with anti-CD3 (145-2C11) and protein A agarosebeads. Immunoprecipitates were eluted from the protein A agarose innon-reducing sample buffer (containing 2% SDS) and were either directlyelectrophoresed or reimmunoprecipitated with specific anti-TcR seraprior electrophoresis. The generation of the anti-TcR antisera has beendescribed previously (53). Analyses were performed on2-dimensional/off-diagonal (2-D) gels (54). Lysates were first subjectto electrophoresis under non-reducing conditions in 10% polyacrylamideSDS-PAGE tube gels and were then electrophoresed under reducingconditions (5% 2-ME) on 10% polyacrylamide SDS-PAGE slab gels. Proteinswere visualized by autoradiography. Molecular mass determinations weremade by comparison to pre-stained high molecular mass standard markers.

EXAMPLE 16 Coupling the Immunopotentiating Protein to the Second Proteinto form a Heteroconjugate

For purposes of this invention, any of the various procedures availableto form cross-links between proteins can be utilized. The method chosenmust not destroy the immunopotentiating activity of the heteroconjugate.Several possible methods are presented below:

A. Biotin-Avidin Coupling

In this method, both of the proteins to be linked are individuallycoupled to biotin molecules. Avidin then forms a bridge between thebiotin molecules, thereby linking the two proteins. This is not acovalent bonding. A variation of the biotin-avidin method is to coupleone protein to biotin, the other to avidin, and to mix the two compoundsresulting in biotin-avidin links. (41)

B. Coupling Effected by Heterobifunctional Reagents

Whole antibodies or fragments are crosslinked using theheterobifunctional reagent succinimidyl-3-(2-pyridyldithiol) propionate(SPDP) at a 3-fold molar excess SPDP to protein. After crosslinking, asmall amount of iodoacetamide is added each sample is applied to a1.6×90 cm Ultragel AcA 22 (LKB Instruments, Inc., Gaithersburg, Md.)column to remove uncoupled monomers. Functional groups are introduced oneach of the proteins to be conjugated so that the predominant reactionwhen the two substituted proteins are mixed, is between the introducedfunctional groups. A preferred reagent used for this purpose isN-succinimidyl-3-(2-pyridyldithio) propionate. Disulfide groups areintroduced into one of the proteins to be conjugated, and thiol groupsare introduced in the other by pyridyldithiolation and subsequentreduction with dithiothreitol. Upon being mixed, the two substitutedproteins conjugate by means of a disulfide bond. The details of thismethod are provided by Carlsson (42). This method has been successfullyapplied to form antibody-toxin conjugates (43).

C. Cross-Linking of Maleimide and SH Groups

SH groups on an one protein are fully alkytated withO-phenylenedimaleimide to provide free maleimide groups. The otherprotein is reduced to produce SH groups. The two preparations are thencross-linked (44).

D. Peptide-Antibody Coupling with Sulfosuccinimidyl4-(N-Maleimidomethyl) Cyclohexane-1-Carboxylate (“Sulfo-SMC”):

Materials:

-   -   1. Antibody to be coupled.    -   2. Peptide to be coupled.    -   3. Dithiothreitol    -   4. Sulfo-SMCC (Pierce 22322)    -   5. Degassed PBS, pH 7.2.    -   6. Dialysis tubing with 12,000-14,000 NW cutoff (Spectra/Por        08-667A).    -   7. SEP-PAK C18 cartridges (Waters 51910).    -   8. Acetonitrile

Procedure:

-   -   1. Dialyse 10 mg antibody into 10 ml PBS pH 7.2; degas buffer        with vacuum prior to use.    -   2. Add 10-fold molar excess Sulfo-SMCC and stir for two hours at        room temperature.    -   3. Remove excess Sulfo-SMCC by dialysing against degassed PBS.    -   4. Reduce peptide with 100 mM dithiothreitol for one hour at 37        degrees.    -   5. Remove excess reductant with SEP-PAK C18 cartridge; prewet        cartridge with degassed PBS, run reduced peptide over cartridge,        and wash with degassed PBS; the reduced peptide will be        retained.    -   6. Elute with 1 ml acetonitrile directly into derivatized        antibody while stirring.    -   7. Incubate for 20 hours at 4 degrees.    -   8. Remove uncoupled peptide by dialysing against PBS.        E. Peptide-Antibody Coupling with 2.2′-Dipyridyl Dithiol (2-PD):

Materials:

-   -   1. Antibody to be coupled.    -   2. Peptide to be coupled.    -   3. Dithiothreitol    -   4. 2-PD (Aldrich 14,309-9; “Aldrithiol-2”)    -   5. Sephadex G25 column (Pharmacia 17-0851-01)    -   6. Degassed PBS, pH 7.2 with EDTA 1 mM.    -   7. Dialysis tubing with 1000 NW cutoff (Spectra/Por 131084,        Spectrum Medical Industries) and with 12,000-14,000 cutoff        (Spectra/Por 08-667a).

Procedure:

-   -   1. Dialyse antibody into PBS pH 7.2+EDTA 1 mM; degas buffer with        vacuum prior to use.    -   2. Reduce with 250-fold molar excess of dithiothreitol for 1.5        hours at room temperature.    -   3. Remove excess reductant with Sephadex G25 sizing column (or        by dialysis).    -   4. Add 2-PD in 100-fold molar excess to block newly exposed        sulfhydryl groups and monitor reaction by OD at 380 nm or        visually by appearance of orange color.    -   5. Remove excess 2-PD with G25 column (or by dialysis).    -   6. Concentrate antibody to 10-15 mg/ml.    -   7. Reduce peptide with DTT as above.    -   8. Remove reductant from peptide by dialysing against degassed        PBS, pH 7.2+1 mM EDTA using 1000 MW cutoff dialysis tubing.    -   9. Add 5 to 10-fold molar excess of reduced peptide to        concentrated antibody; keep total volume small so that the final        concentration of antibody is at least 10 mg/ml.    -   10. Monitor reaction as above.    -   11. Remove uncoupled peptide by dialysing with standard (MW        cutoff 12,000-14,000) dialysis tubing.

Other Methods EXAMPLE 17 Cell Separation and Tissue Culture Techniques

General techniques for purifying T cells by cell sorting or antibody andcomplement treatment routinely performed by published laboratory methods(49) well-known to those skilled in the art.

EXAMPLE 18 Preparation of a Vaccine

All immune responses are dependent on the ability of T cell to recognizeprocessed antigen associated with major histocompatibility antigens(MHC). Any vaccine approach which utilizes, e.g. HIV peptides orinactivated virus antigen must depend on the ability of antigenicpeptides to bind the appropriate MHC antigens necessary to initiate animmune response. Given the tremendous polymorphism of the MHC antigensexpressed in the population and the variation of the virus, developing asuccessful HIV vaccine for general use is difficult.

The efficacy of vaccines, in particular those which may be weaklyimmunogenic, may be improved by modifying the foreign antigen such thatit is more immunogenic, or allowing the use of peptides which are notimmunogenic under normal conditions, since they would not bind MHC, butwhich may constitute a conserved site on the major HIV viral proteins.

Mice are immunized with an amount of heteroconjugate between the amountsof 100 ug and 5 mg added to 30 μl-1 ml of a physiologic salt solution orFreund's adjuvant (Difco). Alum or pertussis may be added as adjuvants.This solution is then injected into a mammal (mouse, rat, hamster,rabbit) or human to stimulate an immune response against the secondprotein. Injection may be subcutaneous, or by an intravenous orintramuscular route. Preferred sites for mice are the foot pad or thebase of the tail. A booster shot is given 30 days after the primaryinjection.

To determine if immunity has been successfully induced, delayed skinhypersensitivity or direct tests for antibodies to the amino acidsequence in the second protein of the heteroconjugate, may be performed.

EXAMPLE 19 Synthetic Peptides

Synthetic peptides corresponding to selected sites are prepared usingstandard methods of solid-phase peptide synthesis on a Vega 250 peptidesynthesizer using double dicyclohexylcarbodimide-mediated couplings (57,58) and butyloxycarbonyl (Boc)-protected amino acid derivatives.Hydroxybenzotriazole preactivation couplings are performed when couplingglutamine or asparagine. The extent of coupling is monitored using thequalitative ninhydrin test and recoupling is performed when <99.4%coupling is observed. Peptides are cleaved from the resin using thelow/high hydrogen fluoride (HF) method (59). For peptide env T2,standard HF cleavage is employed as removal of the tryptophan formylprotecting group is found not to be required for antigenic activity.Peptides are purified to homogeneity by gel filtration and reverse phaseHPLC (60). Composition was confirmed and concentration determined byamino acid analysis (61).

EXAMPLE 20 Purified and Recombinant Proteins

As an example of a protein relevant to HIV, native gp120 is purifiedfrom virus-infected cells as described (62). The recombination proteinsR10 and PB1 are produced by cloning restriction fragments Kpn I(nucleotide 5923) to Bgl II (nucleotide 7197) or Pvu II (nucleotide6659) to Bgl II (nucleotide 7197) from the BH10 clone of type III humanT-cell lymphotropic virus (HTLV-IIIg) into the Repugen expressionvector, followed by expression in Escherichia coli and purification asdescribed (63). R10 is initially solubilized in 20 mM Tris-HCl, pH 8.0,with 10 mM 2-mercaptoethanol at 0.22-0.66 mg/ml. B1 is solubilized in 8M urea at 1.9 mg/ml. Protein R10 represents residues 49-474 of theHTLV-IIIg envelope protein with 25 non-HTLV-III vector-derived residuesat the N terminus and 440 such residues at the C terminus. Protein PB1represents residues 294-474 of gp120 with 30 and 24 non-HTLV-IIIresidues at the N and C termini, respectively. The N-terminal residuesof PB1 are partially shared with those of R10, whereas the C-terminalresidues are unrelated.

Other desired proteins may be prepared by methods well to those skilledin the art.

EXAMPLE 21 Models to Test Use of Anti-CD3 as an Immunopotentiating Agentin Anti-Viral and Anti-Tumor Immunity

A preclinical huSCID model for an anti-human CD3 and staphylococcusenterotoxin augmented viral and tumor immunity may be developed as amodel for the human counterpart, OKT3, or SEB to activate immunity in asimilar manner. Initial studies suggested that patients treated withOKT3 develop activated T cells soon after antibody injection. Inaddition, in other studies, serum from patients have been shown tocontain soon after OKT3 injection various lymphokines and cytokinesincluding TNF, α-interferon, IL-2, and GM-CSF. Therefore, likeanti-murine CD3, OKT3 activates T cells in vivo. However, to determinewhether in vivo activation of human T cells can lead toimmunopotentiation, a model may be developed to examine the OKT3antibody. SCID mice populated with human T and B cells may be used tostudy whether OKT3 will potentiate anti-tumor immune responses.

Approaches:

a: Development of the huSCID model. Severe combined immunodeficiency(SCID) mice have been developed by Bosma, et al. [49]. These micecontain a genetic defect in a recombinase gene which prevents theexpression of mature T cells and B cells. The resulting immunodeficiencyallows for the survival of human cells in these mice. One model forstudying the function of human T and B cells in reconstituted SCID mice(huSCID) has been developed by Mosier, et al. [50]. In this model, 10⁷human peripheral blood cells are injected intraperitoneally into SCIDmice. Four to six weeks post-injection the lymphoid tissues andperitoneal cavity are examined for the presence of human T cells and Bcells. In this model, mice repopulated with human cells have been shownto produce antibodies against diphtheria toxin. Preliminary studies haveshown that SCID mice can be reconstituted with human peripheral blood Tand B cells as monitored in the peritoneal cavity three to six weekspost-injection. In addition, SCID mice reconstituted with HLApresensitized PBL can reject human skin allografts.

b: Treatment of huSCID mice with OKT3 and tumors. After the huSCID modelhas been developed, graded doses of OKT3 were injected into engraftedanimals. Flow cytometry analysis were used to determine the degree of Tcell receptor modulation, depletion, and activation as assessed by IL-2receptor expression. The serum of treated animals were also examined forthe presence of lymphokines and cytokines.

Whereas the invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described in detail. It shouldbe understood, however, that it is not intended to limit the inventionto the particular forms disclosed, but on the contrary, the intention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

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The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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1. An immunopotentiating composition comprising: (a) animmunopotentiating protein; and (b) a second compound having an epitopeagainst which a cellular or humoral immune response is desired.
 2. Thecomposition of claim 1, wherein the immunopotentiating protein comprisesa protein derived from microorganisms.
 3. The composition of claim 2,wherein the protein derived from microorganisms comprises a bacterialprotein.
 4. The composition of claim 3, wherein the bacterial proteincomprises a staphylococcal enterotoxin.
 5. The composition of claim 1,wherein the immunopotentiating protein comprises a monoclonal antibodydirected against a T cell activation molecule on the cell surface of a Tcell.
 6. The composition of claim 5, wherein the T cell activationmolecule comprises a variable or constant region epitope expressed on anantigen specific T cell receptor polymorphic TcR α, β, β, or δ chain. 7.The composition of claim 5, wherein the monoclonal antibody is directedagainst non-polymorphic TcR-associated CD3 chains, γ, δ, or ζ.
 8. Thecomposition of claim 7, wherein the monoclonal antibody comprises OKT3,SP34, or 64.1.
 9. The composition of claim 5, wherein the monoclonalantibody is directed against T cell surface antigens distinct from, andnot physically associated on the cell surface with, TcR.
 10. Thecomposition of claim 9, wherein the monoclonal antibody is directedagainst Thy-1.
 11. The composition of claim 9, wherein the monoclonalantibody is directed against an activation epitope expressed on a memberof the Ly-6 protein family.
 12. The composition of claim 9, wherein themonoclonal antibody(s) is directed against human CD2.
 13. Thecomposition of claim 9, wherein the monoclonal antibody is directedagainst CD28.
 14. The composition of claim 1, wherein theimmunopotentiating protein is a bispecific agent, wherein one arm isspecific for a T cell activation epitope, and the other arm specific fora T cell subset specific epitope.
 15. The composition of claim 14,wherein the bispecific agent comprises a union of two monoclonalantibodies one directed individually against CD3, the other against CD4.16. The composition of claim 1, wherein the second protein comprises apeptide of from about 8 to about 100 amino acids in length.
 17. Thecomposition of claim 1, wherein the second protein comprises a peptideof from about 8 to about 50 amino acids in length.
 18. The compositionof claim 1, wherein the second protein comprises a peptide derived froma tumor-specific or tumor-associated epitope.
 19. The composition ofclaim 1, wherein the second protein comprises a peptide derived from aviral-specific or viral-associated epitope.
 20. The composition of claim1, wherein the second protein comprises a peptide with an amino acidsequence homologous to that derived from a gene in a bacteria, fungus,protozoal or metazoal parasite. 21.-54. (canceled)